Composition for treating HPV (human papillomavirus) infection, nanoparticles and drug system

Abstract

The invention belongs to the technical field of biotechnology and pharmaceutical preparations, and particularly relates to a composition, nanoparticles, a medicine and a medicine system for treating HPV infection. The composition for resisting HPV infection comprises lactoglobulin coupled with a combined antibody and anti-cervicitis pathogen IgY (Immunoglobulin Y), based on the three-in-one antibody combination, the curative effect of immunotherapy can be remarkably improved, the actual curative effect of the combined preparation is greatly improved through the synergistic effect of the three antibodies, the treatment effect of HPV infection is effectively improved, and the combined preparation can be used for preparing drugs and drug systems for resisting HPV infection.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biotechnology and pharmaceutical preparations, and particularly relates to the application of a lactoglobulin conjugated with an antibody and its nanoparticle carrier in combination with an optoelectronic self-controlled cervical drug delivery manipulator. Background Art

[0002] Human papillomavirus (HPV) belongs to the genus Papillomavirus in the family Papovaviridae, and is a circular double-stranded DNA virus. HPV mainly infects the stratified squamous epithelium of the skin and mucous membranes at specific parts of the human body, and sexual contact is its main route of transmission. Other routes include contact transmission or direct mother-to-child transmission. It is reported that women can be repeatedly infected with HPV, or can be infected with multiple different types of HPV at the same time.

[0003] A research report on the analysis of the detection results of human papillomavirus in the female reproductive tract of 3381 cases in China shows that the positive rate of HPV in 3381 women is as high as 18.75%. At the 14th China Summit on Female Reproductive Tract Infections in 2021, some scholars pointed out that the average infection rate of HPV in cytologically normal women globally is estimated to be about 11.7%, and the overall infection rate of high-risk HPV in Chinese women is about 15%. A large number of detection data show that HPV infection is most common among young people aged 18 to 28, and up to 80% of women have had HPV infection before the age of 50, which is a shocking figure.

[0004] Cervical cancer is a disease that seriously endangers the health and life of women, and is also the most common disease related to HPV so far. Cervical cancer occurs in the cervix of women (the entrance from the vagina to the uterus), and is the fourth most common cancer in women. Modern medicine has confirmed that cervical cancer originates from an HPV infection, which is the only cancer with a clear cause among all human cancers currently.

[0005] In addition, cervical inflammation is also a common lower genital tract inflammation in gynecology, accounting for about 40% - 50% of the total number of gynecological outpatient clinics. The epithelium of the endocervical canal is simple columnar epithelium, with many mucosal folds, which is easy to be infected and persistent. The pathogens of chronic cervicitis can ascend to cause endometritis, and can also spread through the lymphatic vessels of the parametrium ligament to cause chronic pelvic inflammatory disease, resulting in abnormal leucorrhea; when the inflammation involves the trigone area of the bladder, it can cause diseases of the urinary system and present irritation symptoms such as dysuria, frequent urination or difficulty in urination. If not diagnosed and treated correctly in time, it can cause some serious diseases, such as infertility, ectopic pregnancy, miscarriage, premature birth, intrauterine fetal death and other complications, and can also cause cervical HPV infection and its related lesions, affecting the quality of life of patients and family harmony.

[0006] So far, the medical community has not developed effective antiviral drugs for the treatment of HPV infections. Traditional antibiotics, germicidal drugs, and disinfectants have no effect on HPV at all. The currently available HPV vaccines usually only have a preventive effect. Although the internationally promoted prophylactic bivalent, quadrivalent, and nonavalent vaccines can prevent 2, 4, and 9 types of human papillomavirus (HPV) infections respectively, they do not have a therapeutic effect, and injecting such vaccines into people already infected with HPV is ineffective. What's more troublesome is that there are more than 100 types of human papillomavirus (HPV), and different types cause different clinical manifestations. The human papillomavirus (HPV) prevalent in different countries and regions is also different. Even vaccine recipients may still be infected with other types of human papillomavirus not covered by the vaccinated vaccine, and it is simply impossible to achieve the goal of completely preventing different human papillomavirus (HPV) infections in different regions by simply increasing the types of vaccine target antigens.

[0007] Currently, in addition to taking various chemical drugs, traditional Chinese medicines, and biological agents orally and by injection, various methods are generally used to perfuse or pack drugs and biological agents into the vagina in order to achieve the purpose of preventing and treating HPV infections, as well as treating cervical cancer and cervicitis. Through in-depth research, it has been found that the cervix is the main site where human papillomavirus (HPV) and cervicitis pathogenic bacteria gather and multiply, and it is also the primary site of cervical cell lesions and cervical cancer. To turn positive patients infected with HPV negative, it is necessary to remove the human papillomavirus hidden in the cervix and the infected cells in the cervix. However, due to the innate physiological characteristics of women, the cervical os is usually in a closed state, and the drug delivery methods of vaginal perfusion or packing cannot deliver drugs and biological agents to the cervical position. As a result, the drugs will inevitably fail to reach the lesion site to take effect, leading to poor actual preventive and therapeutic effects, and the actual curative effects of various drugs for turning positive are very poor. Although the drug can be placed in the cervix by performing a cervical dilation operation, this requires an operation in a specialized medical institution, which is not only time-consuming and laborious, but also increases the cost and pain of the cervical dilation operation, and privacy cannot be guaranteed, reducing the compliance of patients and making it difficult to promote and apply. In addition, the currently widely used chemical drugs and traditional Chinese medicines themselves do not have an ideal therapeutic effect on such diseases, while other physical therapies such as laser and cryotherapy may cause intraoperative bleeding, postoperative cervical canal stenosis, adhesions, infertility, infections, etc., and induce various complications.

[0008] Therefore, the field expects to develop a more effective pharmaceutical composition for the treatment of HPV infections, especially a drug system that can more effectively deliver drugs directly to the cervical region to achieve precise and efficient preventive and therapeutic effects. Summary of the Invention

[0009] To this end, the technical problem to be solved by the present invention is to provide a composition for anti-HPV infection and cervicitis, the composition comprising lactoglobulin conjugated with a combined antibody and anti-cervicitis pathogen IgY, which can effectively prevent and treat diseases such as HPV infection and cervicitis;

[0010] The second technical problem to be solved by the present invention is to provide a nanoparticle preparation based on lactoglobulin conjugated with a combined antibody and anti-cervicitis pathogen IgY;

[0011] The third technical problem to be solved by the present invention is to provide an external preparation for anti-HPV infection based on lactoglobulin conjugated with a combined antibody and anti-cervicitis pathogen IgY;

[0012] The fourth technical problem to be solved by the present invention is to provide a drug system that realizes better drug administration application based on an optoelectronic self-controlled cervical drug delivery manipulator.

[0013] To solve the above technical problems, a composition for anti-HPV infection and cervicitis according to the present invention, the composition comprising lactoglobulin conjugated with a combined antibody and anti-cervicitis pathogen IgY;

[0014] Preferably, the mass ratio of lactoglobulin conjugated with a combined antibody to anti-cervicitis pathogen IgY is (1 - 10) : (1 - 10).

[0015] Specifically, in the composition for anti-HPV infection, in the lactoglobulin conjugated with a combined antibody, the combined antibody comprises a mixture of broad-spectrum anti-HPV-IgY and its small molecule antibody Fab, composite anti-E6 / E7-IgY and its small molecule antibody Fab, and anti-PD-1 / L1-IgY and its small molecule antibody Fab;

[0016] Preferably, the mass ratio of broad-spectrum anti-HPV-IgY, composite anti-E6 / E7-IgY, and anti-PD-1 / L1-IgY is (1 - 10) : (1 - 10) : (1 - 10);

[0017] Preferably, the mass ratio of broad-spectrum anti-HPV-IgY, broad-spectrum anti-HPV-IgY small molecule antibody Fab, composite anti-E6 / E7-IgY, composite anti-E6 / E7-IgY small molecule antibody Fab, anti-PD-1 / L1-IgY, and anti-PD-1 / L1-IgY small molecule antibody Fab is (1 - 10) : (1 - 10) : (1 - 10) : (1 - 10) : (1 - 10) : (1 - 10).

[0018] Specifically, in the composition for anti-HPV infection, the preparation method of the lactoglobulin conjugated with a combined antibody comprises:

[0019] (a) Prepare the required antigens respectively according to the selected antibody types;

[0020] (b) Prepare the corresponding immune eggs respectively using the prepared antigens;

[0021] (c) Prepare the IgY antibodies of the required types and their small molecule antibody Fabs respectively, and obtain the required combined antibodies;

[0022] (d) Couple the combined antibodies with anhydrified lactoglobulin to obtain the product.

[0023] Specifically, the pathogens of the IgY against cervicitis pathogens include the main pathogen types causing vaginitis;

[0024] Preferably, the pathogens include a mixture of Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli, Klebsiella pneumoniae, and Streptococcus agalactiae.

[0025] Specifically, the preparation method of the IgY against cervicitis pathogens includes:

[0026] (a) Take the pathogens of the selected category for culture and prepare the corresponding composite antigen;

[0027] (b) Prepare immune eggs using the composite antigen;

[0028] (c) Prepare the IgY against cervicitis pathogens using the immune eggs to obtain the product.

[0029] The present invention also discloses a nanoparticle against HPV infection, and the nanoparticle includes the composition against HPV infection.

[0030] Specifically, for the nanoparticle against HPV infection, the nanoparticle includes the composition against HPV infection encapsulated by a liposome as a carrier;

[0031] Preferably, the liposome includes at least one of lipid liquid crystal nanoparticles, lipid nanoparticles, or solid lipid nanoparticles.

[0032] The present invention also discloses an external preparation against HPV infection, including the composition against HPV infection and / or the nanoparticle against HPV infection, and acceptable excipients.

[0033] Specifically, for the external preparation, the preparation includes a gel, a dressing, a spray, a gynecological lotion, a men's lotion, a hand sanitizer, a powder, a tablet, a toothpaste, an oral paste, a mouthwash, a buccal tablet, an oral liquid, an oral preparation, or a capsule.

[0034] The present invention also discloses a drug system against HPV infection, including the external preparation against HPV infection, and an optoelectronic self-controlled cervical drug delivery manipulator.

[0035] Specifically, for the described drug system, the optoelectronic self-controlled cervical drug delivery manipulator includes:

[0036] A robotic arm having a first chute and a second chute. An opening is provided at the head of the first chute, and a push rod is arranged in the first chute. The push rod is adapted to extrude the drug from the opening of the first chute. A transparent photoelectric eye is hermetically arranged at the head of the second chute;

[0037] Robotic fingers are arranged at the head of the first chute, and liquid outlets are arranged on the outer surface of the robotic fingers;

[0038] A light-emitting electronic eye visual component is inserted and arranged in the second chute. The light-emitting electronic eye visual component has a camera and a lighting lamp extending towards the head of the second chute.

[0039] Optionally, a plurality of the liquid outlets are evenly arranged along the axis of the robotic fingers.

[0040] Optionally, a syringe is arranged in the first chute. The syringe has an injection tube for accommodating the drug. The push rod is inserted into the injection tube. An injection port for extruding the drug is provided at the head of the syringe, and the robotic fingers are communicated with the injection port.

[0041] Optionally, a connecting sleeve is also included, which is inserted through the first chute. The head of the connecting sleeve is connected to the robotic fingers, and the outer wall of the syringe is inserted into the connecting sleeve.

[0042] Optionally, the robotic fingers are made of a flexible material and are bent. The syringe or the connecting sleeve slides in the first chute, driving the robotic fingers to retract or extend out of the first chute; the robotic fingers with a flexible bent shape are squeezed by the vagina or the first chute, resulting in a change in the bending angle. By adjusting the position of the robotic fingers in the vagina or the first chute, the bending angle of the robotic fingers is changed.

[0043] Optionally, a first clamping groove is provided at the head of the first chute of the robotic arm or the connecting sleeve or the syringe. A tapered chuck with reinforcing ribs is provided at the root of the robotic fingers. The reinforcing ribs of the tapered chuck are clamped into the first clamping groove, so that the robotic fingers are connected to the first chute of the robotic arm or the connecting sleeve or the syringe.

[0044] Optionally, the tail of the connecting sleeve extends towards the outside of the robotic arm, and a handle is provided at the tail of the connecting sleeve.

[0045] Optionally, a second clamping groove is provided at the handle, and the shoulder of the syringe is clamped into the second clamping groove.

[0046] Optionally, a handle is provided on the shoulder of the syringe.

[0047] Optionally, a disc is provided at the tail of the robotic arm, a positioning groove is provided on the disc, and dedicated snap structures are provided on the connecting sleeve, the dedicated injection assembly, and the visible light emitting electronic eye assembly. After the snap structures are aligned with the positioning groove, they are rotated and locked.

[0048] Optionally, the head of the second chute is provided with an extending edge extending outward, and the extending edge is provided on the outer edge of the transparent electric eye.

[0049] Optionally, the visible light emitting electronic eye assembly includes: a camera, a lighting lamp, a control box, and a mounting tube. The control box is provided at the tail of the mounting tube, the camera and the lighting lamp are provided at the head of the mounting tube, and the camera and the lighting lamp extend toward the head of the second chute through the mounting tube; a PCB main board, a gimbal gyroscope, and a switch are provided in the control box.

[0050] The present invention also discloses the use of the anti-HPV infection composition and / or the anti-HPV infection nanoparticles for preparing a drug for preventing and treating HPV infection, cervical infectious diseases or cervical cancer; or,

[0051] The use of the anti-HPV infection composition and / or the anti-HPV infection nanoparticles for preparing a non-diagnostic test reagent or test product; or,

[0052] The use of the anti-HPV infection composition and / or the anti-HPV infection nanoparticles for preparing a medical device for anti-HPV infection.

[0053] The anti-HPV infection composition described in the present invention specifically includes: broad-spectrum anti-HPV-IgY and its small molecule antibody Fab, anti-HPV-E6 / E7-IgY and its small molecule antibody Fab, and anti-PD-1 / L1-IgY and its small molecule antibody Fab; among them, the broad-spectrum anti-HPV-IgY and its small molecule antibody Fab can bind to various subtypes of HPV and have a much stronger binding force to HPV than mammalian antibody IgG; anti-HPV-E6 / E7-IgY can most effectively inhibit the oncogene proteins E6 / E7, and only chicken antibody (IgY) can specifically bind to and inhibit the HPV-E7 oncoprotein in positive cells and in diseased squamous epithelia; anti-PD-1 / L1-IgY is innovated based on the international Nobel Medicine Prize technology and can restart the human immune mechanism and activate T cells to produce a long-term effect. The anti-HPV infection composition described in the present invention can significantly improve the efficacy of immunotherapy based on the "three-in-one" antibody combination. The three antibodies act synergistically, greatly enhancing the actual efficacy of the combined preparation of the present invention. Compared with the efficacy produced by injecting PD-1 inhibitors (such as atezolizumab, durvalumab, and avelumab, etc.) and transporting antibodies to the cervix through blood circulation, the efficacy is greatly improved.

[0054] β-lactoglobulin in the anti-HPV infection composition described in the present invention binds to negatively charged acid anhydride-modified β-lactoglobulin and positively charged HPV to form a protein complex, reducing the HPV viral load. Coupling acid anhydride-modified β-lactoglobulin with the combined antibody can effectively increase the ability of acid anhydride-modified β-lactoglobulin to inhibit the E6 / E7 oncogenes, endowing acid anhydride-modified β-lactoglobulin with a new pharmacological effect, and taking advantage of the respective advantages of acid anhydride-modified β-lactoglobulin and the combined antibody to produce a superimposed effect and improve the therapeutic effect.

[0055] The anti-HPV infection nanoparticles described in the present invention are encapsulated based on advanced carriers such as lipid liquid crystalline nanoparticles (LLCN), Lipid Nanoparticle (LNP), and Solid lipid nanoparticle (SLN), which can significantly improve the ability of the combined antibody to penetrate into the cervical crypts and mucosal spaces, and at the same time endow tissue targeting characteristics, enabling the combined antibody to accurately target and accumulate in the cervical mucosal layer and slowly release the drug; thus giving full play to the efficacy of the nanoparticle preparation of the coupled combined antibody of lactoglobulin and anti-cervicitis pathogen IgY and improving the therapeutic effect.

[0056] The drug system for anti-HPV infection described in the present invention is directly administered into the cervix through an optoelectronic self-controlled cervical drug delivery manipulator, directly entering the cervix to eliminate human papillomavirus and cervicitis pathogens, solving the problem that the drug delivery methods of vaginal perfusion or packing cannot deliver drugs and biological agents to the cervix because the cervix is usually in a closed state (this is an inherent physiological characteristic of women), and greatly improving the curative effect. It can not only significantly increase the positive-to-negative conversion rate, breaking through the restricted area that the negative conversion rate cannot exceed 90%, but also greatly shorten the treatment cycle, shortening the positive-to-negative conversion time to within one month (the medication cycle of currently marketed products is approximately 3 months, similar to the natural apoptosis cycle of infected cells' metabolism), enabling patients to quickly get rid of the distress of painful treatment and get out of the shadow of cervical cancer earlier.

[0057] The drug system for anti-HPV infection described in the present invention combines the three technologies of light, electricity, and machinery skillfully with the optoelectronic self-controlled cervical drug delivery manipulator. Patients can operate it simply and autonomously at home, saving time, effort, and expenses, bringing good news to women around the world and having great social significance.

[0058] The optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system described in the present invention slowly inserts the robotic arm into the vagina. The transparent electro-optical eyes sealed at the head of the second chute protect the camera and the illuminating lamp. Through the camera and the illuminating lamp extending towards the head of the second chute by the light-emitting electro-optical eye visual component, in addition to being able to observe the mechanical finger part and guide the mechanical finger to find the cervical orifice, it can also directly understand the pathological changes of the vagina and the surrounding tissues of the cervical orifice; after the mechanical finger reaches the part where spraying is required, the push rod is pressed to inject the liquid medicine from the liquid outlet on the outer surface of the mechanical finger into the cervix. The optoelectronic self-controlled cervical drug delivery manipulator provided by the present invention combines the three technologies of light, electricity, and machinery skillfully, and can directly deliver drugs and various preparations to the cervical part independently, achieving accurate and efficient prevention and treatment effects, solving the problem in the prior art that drugs cannot be accurately delivered to the lesion site of the cervix independently; at the same time, it can monitor the curative effect and can be used for home health care and gynecological health examinations.

[0059] The optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system described in the present invention has a plurality of liquid outlets evenly arranged on the surface of the mechanical finger, which can spray the drug circumferentially in the cervix, enabling the drug to cover as many lesion sites in the cervix as possible.

[0060] When in use, the optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system described in the present invention inserts the syringe with the drug into the first chute, connects the mechanical finger with the injection port of the syringe, pushes the push rod, and the drug is extruded from the liquid outlet of the mechanical finger. After the drug is used up, the syringe can be withdrawn to clean and disinfect the robotic arm, facilitating the recycling of the robotic arm.

[0061] The optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention has a mechanical finger connected to the head of a connecting sleeve. A syringe is inserted into the connecting sleeve from the tail of the connecting sleeve. The head of the syringe is movably connected to the tail of the mechanical finger, so that the injection port of the syringe communicates with the mechanical finger and is installed in place. By sliding the connecting sleeve, the mechanical finger is driven to retract or extend.

[0062] When the syringe or the connecting sleeve connected to the mechanical finger of the optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention slides in the first chute, the mechanical finger is driven to retract or extend from the first chute. The flexible and curved mechanical finger is squeezed by the vagina or the first chute, resulting in a change in the bending angle. By adjusting the position of the mechanical finger in the vagina or the first chute, the bending angle of the mechanical finger is changed.

[0063] For the optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention, the conical chuck of the mechanical finger is inserted into the first chute of the robotic arm or the first card slot of the connecting sleeve or the head of the syringe, so that the mechanical finger is connected to the first chute of the robotic arm or the connecting sleeve or the syringe. Through the engagement of the reinforcing rib of the conical chuck and the bayonet of the first card slot, the angle of the mechanical finger is fixed.

[0064] For the optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention, the tail of the connecting sleeve extends to the outside of the robotic arm. By means of the handle at the tail of the connecting sleeve, the connecting sleeve is slid along the first chute, thereby driving the mechanical finger to extend or retract from the first chute and adjusting the angle of the mechanical finger.

[0065] For the optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention, it is fixed by relying on the shoulder of the syringe being clamped into the second card slot at the handle, preventing the syringe from slipping out, and enabling the head of the syringe to be tightly pressed against the root of the mechanical finger to prevent drug leakage.

[0066] For the optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention, by means of the handle provided on the shoulder, the syringe is rotated so that the shoulder of the syringe is clamped into the second card slot at the handle, which is convenient for operation.

[0067] For the electro-self-controlled cervical drug delivery manipulator provided by the drug system of the present invention, the disc at the tail of the robotic arm facilitates the user to hold the robotic arm during operation. After aligning with the positioning slot through a special snap structure, it is rotated and locked to prevent the syringe, the connecting sleeve, the special injection component, and the light-emitting electronic eye visual component from sliding out of the robotic arm during use.

[0068] The optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention uses the protruding edge provided at the head of the second chute to support the side wall tissues of the vagina and cervix, preventing the side wall tissues of the vagina and cervix from covering the transparent photoelectric eye and ensuring the visibility of the field of view; it can also enter the cervical canal to observe the lesion conditions and monitor the quality effects.

[0069] For the optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention, the camera and the lighting lamp of the light-emitting electronic eye visual component extend towards the head of the second chute through the installation pipe. In addition to being able to observe the mechanical finger part and guide the mechanical finger to find the cervical orifice, it can also directly understand the lesion conditions of the tissues around the vagina and cervical orifice; the control box is arranged at the tail of the installation pipe, and the connecting wires between the camera, the lighting lamp and the control box can be arranged inside the installation pipe, so that the control box is arranged outside the robotic arm, facilitating the control of the light-emitting electronic eye visual component; the light-emitting electronic eye visual component is controlled by the PCB main board and the universal gyroscope box switch in the control box. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in conjunction with the drawings, wherein,

[0071] Figure 1 is a schematic diagram of the first embodiment of the optoelectronic self-controlled cervical drug delivery manipulator provided by the present invention;

[0072] Figure 2 is Figure 1 a schematic diagram of the robotic arm in

[0073] Figure 3 is Figure 2 a schematic diagram of another perspective of

[0074] Figure 4 is Figure 1 a schematic diagram of the mechanical finger in

[0075] Figure 5 is Figure 4 a bottom view schematic diagram of

[0076] Figure 6 is Figure 1 a schematic diagram of the syringe in

[0077] Figure 7 is Figure 1 a schematic diagram of the light-emitting electronic eye visual component in

[0078] Figure 8 is Figure 1 a schematic diagram of the connecting sleeve in

[0079] Figure 9 isFigure 8 Top view schematic diagram;

[0080] Figure 10 Schematic diagram of the connection between the mechanical finger and the connecting sleeve;

[0081] Figure 11 Schematic diagram of the second embodiment of the optoelectronic automatic control cervical drug delivery manipulator provided by the present invention;

[0082] Figure 12 is Figure 11 Schematic diagram of another special injection component in

[0083] Figure 13 is Figure 11 Schematic diagram of the circular chuck with a handle in

[0084] Figure 14 Schematic diagram of the third embodiment of the optoelectronic automatic control cervical drug delivery manipulator provided by the invention;

[0085] Figure 15 is Figure 14 Schematic diagram of the robotic arm of another embodiment;

[0086] Figure 16 is Figure 15 Schematic diagram of another perspective of

[0087] Explanation of reference numerals:

[0088] 1, robotic arm; 2, syringe; 3, mechanical finger; 4, light-emitting electronic eye visual component; 5, first chute; 6, second chute; 7, push rod; 8, injection port; 9, liquid outlet; 10, transparent electric eye; 11, connecting sleeve; 12, first card slot; 13, conical chuck; 14, handle; 15, second card slot; 16, shoulder support; 17, control box; 18, mounting tube; 19, disc; 20, extending edge; 21, handle; 22, circular chuck. Detailed implementation manners

[0089] Example 1

[0090] This example is used to prepare high-purity broad-spectrum anti-HPV-IgY and its small molecule Fab dry powder.

[0091] 1. Preparation of broad-spectrum HPV antigen

[0092] The broad-spectrum HPV antigen in this example is a recombinant polypeptide protein of the common antigen epitopes shared by all types of HPV.

[0093] Although there are more than 200 types of HPV, according to in-depth research, by using modern instrument equipment and high-end algorithm technology to analyze the internal structures of all HPV viruses, it is found that all types of HPV have a group of common antigenic epitope gene sequences. This common and conserved antigenic epitope gene sequence protein can induce animals to produce broad-spectrum antibodies against all serotypes of HPV. The present invention uses bioinformatics technology to screen out the common antigenic epitopes of L1 proteins of high-risk types such as 16, 58, 18, 52, 33, etc. The amino acid sequences of L1 proteins of these five high-risk types of HPV are compared using the Blast software in NCBI to obtain the common polypeptide fragment sequence, and this common antigenic epitope gene sequence protein polypeptide is synthesized through polypeptide synthesis technology.

[0094] Using the method of the present invention has made great technological progress compared with the previous method of screening out the common antigenic epitopes of L1 proteins of some high-risk types of HPV such as types 16, 58, 18, 52, 33 and preparing the conjugate KLH common antigen synthetic epitope polypeptide antigen component. Using the common antigenic epitope gene sequence protein polypeptide studied by the method of the present invention as the antigen component to prepare anti-HPV-E6 / E7-IgY and its small molecule Fab, the obtained antibodies can not only target the antigenic epitopes of L1 proteins of only five limited HPV subtypes such as types 16, 58, 18, 52, 33, but can target the common antigenic epitopes of HPV-L1 and L2 of all serotypes. Discovering and targeting this antigenic epitope shared by all types of HPV and preparing the corresponding antibodies has very important practical significance. In addition to achieving true broad-spectrum resistance, enabling the finally prepared HPV infection prevention product to prevent infections of various subtypes of HPV, it also greatly simplifies the technological process of antigen preparation and can save a lot of costs for practical applications.

[0095] The specific preparation method is as follows:

[0096] (a) Amino acid sequence of HPV16 L1 protein and single parameter setting

[0097] The amino acid sequence of the HPV16 L1 protein is shown in SEQ ID No.1: mslwlpseat vylppvpvskvvstdeyvar tniyyhagts rllavghpyf pikkpnnnki lvpkvsglqy rvfrihlpdp nkfgfpdtsfynpdtqrlvw acvgvevgrg qplgvgisgh pllnklddte nasayaanag vdnrecismd ykqtqlcligckppigehwg kgspctnvav npgdcpplel intviqdgdm vdtgfgamdf ttlqanksev pldictsickypdyikmvse pygdslffyl rreqmfvrhl fnragavgen vpddlyikgs gstanlassn yfptpsgsmvtsdaqifnkp ywlqraqghn ngicwgnqlf vtvvdttrst nmslcaaist settykntnf keylrhgeeydlqfifqlck itltadvmty ihsmnstiledwnfglqppp ggtledtyrf vtsqaiacqk htppapkedplkkytfwevn lkekfsadld qfplgrkfll qaglkakpkf tlgkrkatpt tsststtakr kkr。

[0098] Using the ExPASy-ProtScale and Antibody epitope prediction antigen epitope prediction websites, four main parameters were selected: hydrophilicity prediction (Hopp&Woods Hydrophilicity Prediction), flexibility prediction (Karplus&Schulz Flexibility Prediction), β-turn prediction (Chou&Fasman Beta-Turn Prediction), and antigen epitope index prediction (Kolaskar&Tongaonkar Antigenicity Prediction). The amino acid sequence of the HPV16 L1 capsid protein was input into the working area, and each of its parameters was analyzed.

[0099] (b) Alignment of common antigen epitopes of high-risk HPV and prediction of spatial structure to obtain the sequence of the common polypeptide fragment

[0100] Using the UniProtKB protein library, with HPV16 L1 as the standard, the highest homology of the L1 proteins of types 58, 18, 52, and 33 with it is 78%, 64%, 77%, and 79% respectively. Then, using the Blast software in NCBI, the amino acid sequences of the L1 proteins of these five high-risk types of HPV were aligned to obtain the common polypeptide fragment sequence. Then, this epitope polypeptide was synthesized through polypeptide synthesis technology.

[0101] (c) To improve the immunogenicity of the protein polypeptide of this common epitope gene sequence, this common epitope polypeptide was conjugated with KLH (or highly immunogenic carrier proteins such as BSA, OVA, etc.) to prepare a common antigen synthetic epitope polypeptide antigen component conjugated with KLH (or highly immunogenic carrier proteins such as BSA, OVA, etc.).

[0102] This common epitope polypeptide was linked to KLH (or highly immunogenic carrier proteins such as BSA, OVA, etc.) through chemical linkage or gene recombination technology. Among them, there are many applicable chemical linkage methods, such as carbodiimide method, glutaraldehyde method, periodate oxidation method, succinic anhydride method, carboxymethylhydroxylamine method, diazotized p-aminobenzoic acid method, sodium monochloroacetate method, active ester method, acid anhydride method, and mixed acid anhydride method, etc. Heterobifunctional reagents such as SPDP can also be used as cross-linking agents for conjugation. The specific process will not be elaborated in this example.

[0103] In this example, the prepared common antigen synthetic epitope polypeptide antigen component conjugated with KLH was mixed with Freund's adjuvant in a ratio of (1 - 10):(1 - 10), and stirred evenly to prepare a broad-spectrum HPV antigen.

[0104] It should be noted that this example only takes the common antigen synthetic epitope polypeptide as an example of the broad-spectrum HPV antigen component, but is not limited to this antigen component and the implementation method steps. In practical applications, the common antigen epitope gene sequences of other representative HPV subtypes (such as one or 2 - 6 or more than six of the models of HPV16, 18, 31, 33, 45, 58, etc.) can be selected, the L1 protein genes of these HPV subtypes can be cloned, and they can be modified using codon optimization strategies. The optimized gene fragments are inserted into the pFastBac Dual vector to obtain a recombinant vector. After transforming Escherichia coli DH10Bac competent cells, a recombinant Bacmid is obtained, which is transfected into insect Sf9 cells. The expression of the recombinant protein is detected by Western blotting and SDS-PAGE. The specific process will not be elaborated in this example.

[0105] 2. Preparation of immune eggs

[0106] Select laying poultry (such as chickens, ducks, geese, turkeys, ostriches, etc.) with high immune response ability, apply the immune activation method, and perform immune injection with the prepared broad-spectrum HPV antigen. Inject the broad-spectrum HPV antigen prepared in the above steps into laying hens every two weeks, and inject 3 times. After 15 - 20 days of the first immunization, collect the immune eggs produced by the laying hens respectively to obtain anti-broad-spectrum HPV-IgY immune eggs.

[0107] 3. Preparation of broad-spectrum anti-HPV-IgY and anti-HPV small molecule antibody Fab

[0108] The method for preparing anti-HPV-IgY in this example can adopt the pure water extraction method, chloroform extraction method, cold ethanol precipitation method or ammonium sulfate precipitation method to prepare the crude extract of broad-spectrum anti-HPV-IgY.

[0109] Taking the way of preparing the crude extract of anti-broad-spectrum HPV-IgY by the pure water extraction method as an example in this example, the specific operations include: washing the prepared broad-spectrum anti-HPV-IgY immune eggs with running water, disinfecting them by wiping with alcohol, then breaking the broad-spectrum anti-HPV-IgY immune eggs with an egg beater, filtering out the egg white with an egg yolk sieve, leaving the egg yolk, and stirring evenly; then adding distilled water at 3 - 8 times the volume of the egg yolk liquid, diluting and mixing evenly, and adjusting the pH to 5.5 - 6.5 with 1.0N HCl solution; further stirring the diluted liquid with adjusted pH evenly, then cooling it to 2 - 6 °C, and standing for 12 - 24 hours; centrifuging the diluted liquid at high speed; taking the supernatant obtained by separation and placing it in an ultrafilter for ultrafiltration and concentration by 10 - 20 times; then adding a sodium alginate solution with a concentration of 1.0 - 3.0%, slowly adding the sodium alginate solution until the final concentration is 0.1 - 0.2%, and stirring until turbidity appears; then adding a 1.0 - 3.0% CaCl2 solution until the final concentration is 0.1 - 0.2%, stirring evenly, and standing at 3 - 4 °C for 8 - 12 hours; centrifuging at high speed and taking the supernatant to obtain the crude extract of anti-broad-spectrum HPV-IgY. Then, passing the crude extract of anti-broad-spectrum HPV-IgY through an ion exchange column and an affinity chromatography column for chromatography, and dialyzing and concentrating the chromatographic collection to obtain a high-purity broad-spectrum anti-HPV-IgY solution.

[0110] Continue to adjust the pH of the crude extract solution of anti-broad-spectrum HPV / L1-IgY to 3.0 - 5.0, add catalytic protease; stir and dissolve fully to produce an enzymatic reaction, then, centrifuge at high speed at low temperature, discard the precipitate to obtain the supernatant, and perform ultrafiltration on the supernatant to obtain a concentrated solution; then pass the concentrated solution obtained after ultrafiltration through an ion exchange column and an affinity chromatography column for chromatography in sequence, and dialyze and concentrate the chromatographic collection to obtain a high-purity broad-spectrum anti-HPV small molecule antibody Fab solution.

[0111] In this embodiment, a bacterial and viral filtration device of a virus removal filtration system manufactured by Pall Ultrafine Filtration Company of the United States is used to thoroughly filter out various bacteria and viruses, ensuring that the prepared IgY and small molecule antibody Fab are absolutely free of any viruses and bacteria. Among them, the first bacterial filtration device uses a 0.22μm membrane sterilization filter to remove bacteria such as Salmonella; the second mycoplasma filtration device uses a 0.1μm membrane mycoplasma removal filter to remove mycoplasma; the third virus filtration device uses an Ultipor VF™ DV50 virus removal filter to remove various viruses including influenza virus and enterovirus.

[0112] After freeze-drying, or spray-drying at medium and low temperature, or fluidized bed drying, or other drying methods that do not affect the antibody activity, the above-prepared high-purity broad-spectrum anti-HPV-IgY solution and broad-spectrum anti-HPV small molecule antibody Fab solution are respectively added to an ultrafine pulverizer for grinding and crushing, and high-purity nano broad-spectrum anti-HPV-IgY dry powder and high-purity nano broad-spectrum anti-HPV small molecule antibody Fab dry powder are respectively prepared.

[0113] Example 2

[0114] This embodiment is used to prepare high-purity broad-spectrum anti-E6 / E7-IgY and its small molecule Fab nano dry powder.

[0115] 1. Preparation of HPV-E6 / E7 antigen components

[0116] In this embodiment, various known methods can be used to prepare the HPV-E6 / E7 antigen component. For example, a recombinant bacterial vector containing modified HPV 16E7 or a recombinant virus expressing HPV-16 / 18 type E6 / E7 can be used, and various synthetic HPV16 E7 polypeptides can also be used. In addition, the E7 proteins of HPV16 and HPV18 can be combined with specific enzymes, and DNA plasmids encoding HPV 16 / 18 type E6 and E7 can be used as antigen components; the specific preparation process follows the conventional steps, and the specific process and method will not be elaborated here.

[0117] Since the E6 / E7 antigen gene structures of different types of HPV are also different, 15 high-risk types of HPV are randomly divided into 5 groups, such as:

[0118] Group A: 16, 18, 58 (the highest infection rate among Chinese women)

[0119] Group B: 33, 52, 31

[0120] Group C: 35, 39, 45

[0121] Group D: 51, 56, 59

[0122] Group E: 68, 73, 82

[0123] The E6 / E7 fusion genes of 15 high-risk HPV virus subtypes are grouped and expressed in eukaryotic cells or prokaryotic cells and then purified to prepare the eukaryotic or prokaryotic expression protein antigen components of the E6 / E7 fusion genes.

[0124] This grouping method is a solution; however, there are three HPV-E6 and three HPV-E7 in each group of three types, a total of six HPV-E6 / E7 gene proteins, and 30 HPV-E6 / E7 gene proteins in 5 groups. This grouping method will inevitably bring a huge workload to antigen preparation.

[0125] Using the method of the present invention, on the basis of epidemiological investigation, the internal structures of 15 high-risk HPVs are analyzed, and four high-risk HPVs 16, 18, 31, and 45 with the greatest harm, the highest incidence, and the most common and representative antigenic epitopes in the internal structure are selected from the 15 high-risk HPVs, and eight polypeptide protein antigen components such as HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV31-E6, HPV31-E7, HPV45-E6, and HPV45-E7 are respectively prepared. The method of the present invention simplifies the production process flow, greatly reduces the actual application cost, and has obvious technological progress. Using these eight most representative polypeptide protein antigen components and their composite antigen components to prepare anti-HPV-E6 / E7-IgY and its small molecule Fab can inhibit more than 90% of the HPV-E6 / E7 oncogenic proteins of subtypes.

[0126] This example takes the preparation of eight representative polypeptide protein antigen components and their composite antigen components as an example, but is not limited to these antigen components and the implementation method steps.

[0127] This example is used to prepare eight representative HPV-E6 / E7 polypeptide protein antigen components.

[0128] (1) HPV16 type-E6 polypeptide protein antigen component

[0129] Based on the HPV16 E6 amino acid sequence shown in SEQ ID No. 2 (GenBank: AKN79013.1): MFPDPQERPI KLPDLCTELP TTIHDIILEC VYCKQQLLRR EVYDFAFRDL CIVYRDGNPY AVCDKCLKFYSKISEYRYYC YSLYGTTLEQ QYNKPLCDLL IRCINCQKPL CPEEKQRHLD KKQRFHNIRG RWTGRCMSCCESSRTRRETQL, using the MOE software, through Modeller homology modeling, the HPV16-type - E6 polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0130] (2) Antigen component of HPV16-type - E7 polypeptide protein

[0131] Based on the amino acid sequence of HPV16 E7 shown in SEQ ID No. 3 (MHGDTPTLHE YMLDLQPETTDLYCYEQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIVCPICSQKPP), using the MOE software, through Modeller homology modeling, the HPV16-type - E7 polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0132] (3) Antigen component of HPV18-type - E6 polypeptide protein

[0133] Based on the amino acid sequence of HPV18 E6 shown in SEQ ID No. 4 (MHGPKATLQD IVLHLEPQNEIPVDLLCHEQ LSDSEEENDE IDGVNHQHLP ARRAEPQRHT MLCMCCKCEARIKLVVESSA DDLRAFQQLFLNTLSFVCPW CASQQ), using the MOE software, through Modeller homology modeling, the HPV18-type - E6 polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0134] (4) Antigen component of HPV18-type - E7 polypeptide protein

[0135] Based on the amino acid sequence of HPV18 E7 shown in SEQ ID No.5 (MHGPKATLQD IVLHLEPQNEPVDLLCHEQ LSDSEEENDE IDGVNHQHLP ARRAEPQRHT MLCMCCKCEA RIELVVESSA DDLRAFQQLFLNTLSFVCPW CASQQ), using the MOE software, through Modeller homology modeling, the HPV18-E6 polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0136] (5) Antigen component of HPV31-E6 polypeptide protein

[0137] Based on the amino acid sequence of HPV31 E6 shown in SEQ ID No.6 (MFKNPAERPRKLHELSSALEIPYDELRLNCVYCKGQLTETEVLDFAFTDLTIVYRDD TPHGVCTKCLRFYSKVSEFRWYRYSVYGTTLEKLTNKGICDLLIRCITCQRPLCPEEKQ RHLDKKKRFHNIGGRWTGRCIACWRRPRTETQV), using the MOE software, through Modeller homology modeling, the HPV31-E6 polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0138] (6) Antigen component of HPV31-E7 polypeptide protein

[0139] Based on the amino acid sequence of HPV31E7 shown in SEQ ID No.7 (MRGETPTLQDYVLDLQPEATDLHCYEQLPDSSDEEDVIDSPAGQAEPDTSNYNIVT FCCQCKSTLRLCVQSTQVDIRILQELLMGSFGIVCPNCSTRL), using the MOE software, through Modeller homology modeling, the HPV31-E6 antigen polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0140] (7) Antigen component of HPV45-E6 polypeptide protein

[0141] According to the amino acid sequence of HPV45 E6 shown in SEQ ID No. 8 (MARFDDPKQRPYKLPDLCTELNTSLQDVSIACVYCKATLERTEVYQFAFKDLCIVY RDCIAYAACHKCIDFYSRIRELRYYSNSVYGETLEKITNTELYNLLIRCLRCQKPLNPAE KRRHLKDKRRFHSIAGQYRGQCNTCCDQARQERLRRRRETQV), using the MOE software, through Modeller homology modeling, the HPV45 type-E6 polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0142] (8) Antigen component of HPV45 type-E7 polypeptide protein

[0143] According to the amino acid sequence of HPV45 E7 shown in SEQ ID No. 9 (MHGPRETLQEIVLHLEPQNELDPVDLLCYEQLSESEEENDEADGVSHAQLPARRAE PQRHKILCVCCKCDGRIELTVESSAEDLRTLQQLFLSTLSFVCPWCATNQ), using the MOE software, through Modeller homology modeling, the HPV45 type-E7 polypeptide protein was constructed, and the specific preparation process will not be elaborated.

[0144] As understood by those skilled in the art, the molecular weights of the polypeptide proteins constructed by the above methods are too small and the immunogenicity is poor. To improve their immunogenicity, they must be conjugated with macromolecular proteins with high immunogenicity, and the specific conjugation process and method can be completed using the methods known in the art.

[0145] Taking the construction and conjugation method of HPV16 type E7 polypeptide protein as an example, this example is described as follows:

[0146] (1) Construction of plasmid

[0147] The E7 gene was amplified by PCR, and the primers used were E7-HPV on the coding strand (5"-GATGCATCACAACATGGAGATACACCTACATTGCAT-3") and E7-HPV on the complementary strand (5"-GGAGCTGTTATGGTTTCTGAGAACAGATGG-3").

[0148] The PCR product was cloned into the pGEM-T Easy Vector to generate the pGEM-E7 plasmid.

[0149] (2) Plasmid expression

[0150] In the specific operation method of this embodiment, plasmid construction is first established in Escherichia coli and then transferred to Lactococcus lactis by electroporation. The nisin induction system is used to express the E7 gene in Lactococcus lactis. The coding gene is ligated to the expression vector and transferred into the recipient cells. After induced expression, the cells are lysed, and the HPV16 E7 polypeptide protein is collected and purified.

[0151] (3) Coupling with highly immunogenic macromolecular proteins

[0152] To improve the immunogenicity of the HPV16 E7 polypeptide protein, the E7 polypeptide protein is coupled with KLH (or other highly immunogenic carrier proteins such as BSA, OVA), and a synthetic antigen component of the polypeptide protein conjugated with KLH (or other highly immunogenic carrier proteins such as BSA, OVA) is prepared. The specific preparation method of the feasibility of this embodiment is as follows:

[0153] A. Dissolve SMCC ((N-Maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester) in 2 ml of DMF (N,N-Dimethylformamide).

[0154] B. Add KLH (keyhole limpet hemocyanin) to a Erlenmeyer flask and supplement with 1×PBS (pH 7.2) to reach the expected final concentration of the carrier protein.

[0155] C. Slowly drip the dissolved SMCC solution into KLH and stir at room temperature for 1 h.

[0156] D. Dialyze with PBS (pH 7.4) solution at 4°C for 6 hours to remove free SMCC.

[0157] E. Pour the dialyzed KLH protein into a centrifuge tube, determine its volume through the scale of the centrifuge tube, calculate the concentration of the protein after dialysis according to the amount of KLH protein added before the reaction, and then transfer the KLH-SMCC solution to the centrifuge tube according to its concentration.

[0158] F. Dissolve the E7 antigen polypeptide protein with PBS (pH 7.2) solution.

[0159] G. Detect the sulfhydryl groups in the polypeptide protein with Ellman's reagent.

[0160] H. Drop the polypeptide protein solution into the KLH-SMCC tube and mix with a vertical mixer at room temperature for 4 hours.

[0161] J. Detection of sulfhydryl groups in polypeptides using Ellman's reagent.

[0162] 2. Preparation of HPV-E6 / E7 composite polypeptide protein antigen

[0163] Respectively mix the previously prepared HPV16 - E6 antigen polypeptide, HPV16 - E7 antigen polypeptide, HPV18 - E6 polypeptide protein, HPV18 - E7 polypeptide protein, HPV6 - E6 polypeptide protein, HPV6 - E7 polypeptide protein, HPV11 - E6 polypeptide protein, and HPV11 - E7 polypeptide protein in a ratio of (1 - 10):(1 - 10):(1 - 10):(1 - 10):(1 - 10):(1 - 10):(1 - 10):(1 - 10) (in this example, the selected ratio is 1:1:1:1:1:1:1:1), and thus the HPV - E6 / E7 composite polypeptide protein antigen component is prepared.

[0164] Mix the prepared HPV - E6 / E7 composite polypeptide protein antigen component with Freund's adjuvant in a ratio of (1 - 10):(1 - 10) (in this example, 1:1), place it in a high - speed homogenizer, and homogenize at a high speed of 8,000 rpm to obtain the HPV - E6 / E7 polypeptide protein composite antigen for immunization.

[0165] 3. Preparation of immune eggs

[0166] Select laying poultry (such as chickens, ducks, geese, turkeys, ostriches, etc.) with high immune response ability, apply the immune activation method, and use the prepared HPV - E6 / E7 polypeptide protein composite antigen for immunization injection. Inject the laying hens once every two weeks for a total of 3 times; 15 - 20 days after the first immunization, respectively collect the immune eggs produced by the laying hens to obtain anti - HPV - E6 / E7 - IgY immune eggs.

[0167] 4. Preparation of anti-HPV-E6 / E7-IgY and anti-HPV-E6 / E7 small molecule antibody Fab

[0168] For the preparation of anti - HPV - E6 / E7 - IgY, a crude extract of anti - HPV - E6 / E7 - IgY can be prepared by the conventional pure water extraction method, chloroform extraction method, cold ethanol precipitation method, or ammonium sulfate precipitation method in this example.

[0169] In this embodiment, the method for preparing the crude extract of anti-HPV-E6 / E7-IgY by the conventional pure water extraction method is taken as an example. The specific operations include: washing the prepared anti-HPV-E6 / E7-IgY immune eggs with running water, disinfecting them by scrubbing with alcohol, then breaking the anti-HPV-E6 / E7-IgY immune eggs with an egg beater, filtering out the egg white with a yolk sieve, leaving the yolk, and stirring evenly; then adding distilled water at 3-8 times the volume of the yolk liquid, diluting and mixing evenly, and adjusting the pH to 5.5-6.5 with 1.0N HCl solution; further stirring the diluted liquid with the adjusted pH value evenly, then cooling it to 2-6°C, and standing for 12-24 hours; centrifuging the diluted liquid at high speed; taking the supernatant obtained by separation and placing it in an ultrafilter for ultrafiltration and concentration by 10-20 times; then adding a sodium alginate solution with a concentration of 1.0-3.0%, slowly adding the sodium alginate solution until the final concentration is 0.1-0.2%, and stirring until turbidity appears; then adding 1.0-3.0% CaCl2 solution until the final concentration is 0.1-0.2%, stirring evenly, and standing at 3-4°C for 8-12 hours; centrifuging at high speed and taking the supernatant to obtain the crude extract of anti-HPV-E6 / E7-IgY. Then, the crude extract of anti-HPV-E6 / E7-IgY is chromatographed through an ion exchange column and an affinity chromatography column, and the chromatographed collected material is dialyzed and concentrated to obtain a high-purity anti-HPV-E6 / E7-IgY solution.

[0170] Continue to adjust the pH of the crude extract solution of anti-HPV-E6 / E7-IgY to 3.0-5.0, and add catalytic protease; stir and dissolve fully to produce an enzymatic reaction, then centrifuge at a high speed with low temperature, discard the precipitate to obtain the supernatant, and perform ultrafiltration on the supernatant to obtain a concentrated solution; then chromatograph the concentrated solution obtained after ultrafiltration through an ion exchange column and an affinity chromatography column in sequence, and dialyze and concentrate the chromatographed collected material to obtain a high-purity anti-HPV-E6 / E7 small molecule antibody Fab solution.

[0171] In this embodiment, a bacterial and viral filtration device of a virus removal filtration system manufactured by Pall Ultrafine Filtration Company of the United States is used to thoroughly filter out various bacteria and viruses to ensure that the prepared IgY and small molecule antibody Fab are absolutely free of any viruses and bacteria. Among them, the first bacterial filtration device is to remove bacteria such as Salmonella with a 0.22μm membrane sterilization filter; the second mycoplasma filtration device is to remove mycoplasma with a 0.1μm membrane mycoplasma removal filter; the third virus filtration device is to remove various viruses including influenza virus and enterovirus with an Ultipor VFTM DV50 virus removal filter.

[0172] After freeze-drying, medium-low temperature spray drying, fluidized bed drying or other drying methods that do not affect the antibody activity of the high-purity anti-HPV-E6 / E7-IgY solution and the anti-HPV-E6 / E7-IgY small molecule antibody Fab solution respectively, they are added to an ultrafine grinder for grinding and pulverization to obtain high-purity nano anti-HPV-E6 / E7-IgY dry powder and high-purity nano anti-HPV-E6 / E7 small molecule antibody Fab dry powder respectively.

[0173] Example 3

[0174] This example is used to prepare high-purity anti-PD-1 / L1-IgY and its small molecule Fab nano dry powder.

[0175] 1. Preparation of PD-1 / L1 composite antigen

[0176] In this example, the human PD-1 and PD-L1 expression proteins are recombinantly expressed respectively using conventional genetic engineering techniques and then mixed in a ratio of (1-10):(1-10) (1:1 is selected in this example); then, it is mixed with Freund's adjuvant in a ratio of (1-10):(1-10) (1:1 is selected in this example) to make a human PD-1 / L1 expression protein complex antigen.

[0177] 2. Preparation of immune eggs

[0178] In this example, laying poultry with high immune response ability (such as chickens, ducks, geese, turkeys, ostriches, etc.) are selected, and the immune activation method is applied to perform immune injection using the prepared human PD-1 / L1 expression protein complex antigen. The laying hens are injected with the human PD-1 / L1 expression protein complex antigen prepared in the above steps every two weeks for 3 times. 15-20 days after the first immunization, the immune eggs produced by the laying hens are collected respectively to obtain anti-PD-1 / L1-IgY immune eggs.

[0179] 3. Preparation of anti-PD-1 / L1-IgY and anti-PD-1 / L1 small molecule antibody Fab

[0180] The preparation of the anti-PD-1 / L1-IgY can adopt the broad-spectrum anti-HPV-IgY crude extract preparation methods known in the art, such as pure water extraction method, chloroform extraction method, cold ethanol precipitation method or ammonium sulfate precipitation method.

[0181] In this embodiment, a pure water extraction method is used to prepare a crude extract of anti-PD-1 / L1-IgY. The specific operation includes: washing the prepared anti-PD-1 / L1-IgY immune eggs with running water, wiping and disinfecting with alcohol, and then breaking the anti-PD-1 / L1-IgY immune eggs with an egg beater, filtering out the egg white with an egg yolk sieve, leaving the egg yolk, and stirring evenly; then adding distilled water at a volume of 3-8 times the volume of the egg yolk liquid, diluting and mixing evenly, and rinsing with 1.0N The pH of the HCI solution is adjusted to 5.5-6.5; the diluted solution with the adjusted pH value is further fully stirred, then cooled to 2-6°C and allowed to stand for 12 hours to 24 hours; the diluted solution is centrifuged at high speed; the separated supernatant is placed in an ultrafilter for ultrafiltration and concentration by 10-20 times; then a sodium alginate solution with a concentration of 1.0-3.0% is added, and the sodium alginate solution is slowly added to a final concentration of 0.1-0.2%, and stirred until turbidity appears; 1.0-3.0% CaCl2 solution is then added to a final concentration of 0.1-0.2%, stirred evenly, and allowed to stand at 3-4°C for 8-12 hours; high-speed centrifugation and the supernatant is taken to obtain a crude anti-PD-1 / L1-IgY extract. The crude anti-PD-1 / L1-IgY extract is chromatographed on an ion exchange column and an affinity chromatography column, and the chromatographic collection is dialyzed and concentrated to obtain a high-purity anti-PD-1 / L1-IgY solution.

[0182] The pH of the anti-PD-1 / L1-IgY solution is adjusted to 3.0-5.0, and a catalytic protein enzyme is added; the solution is fully stirred and dissolved to produce an enzymatic reaction, and then the solution is centrifuged at low temperature and high speed, and the precipitate is discarded to obtain the supernatant, and the supernatant is ultrafiltered to obtain a concentrated solution; the concentrated solution obtained after ultrafiltration is then chromatographed on an ion exchange column and an affinity chromatography column respectively, and the chromatographic collection is dialyzed and concentrated to obtain a high-purity anti-PD-1 / L1 small molecule antibody Fab solution.

[0183] Finally, the bacterial virus filter device of the virus removal filtration system manufactured by Pall Ultrafine Filtration Company of the United States was used to completely filter out various bacteria and viruses, ensuring that the prepared IgY and small molecule antibody Fab do not contain any viruses and bacteria. Among them, the first bacterial filter device uses a 0.22μm membrane sterilization filter to remove bacteria such as Salmonella; the second mycoplasma filter device uses a 0.1μm membrane mycoplasma filter to remove mycoplasma; the third virus filter device uses an Ultipor VFTM DV50 virus filter to remove various viruses including influenza virus and enterovirus.

[0184] The high-purity anti-PD-1 / L1 small molecule antibody Fab solution is freeze-dried or spray-dried at medium and low temperatures or fluidized bed dried or other drying methods that do not affect the antibody activity, and then added to an ultrafine grinder for grinding and pulverization to obtain high-purity nano anti-PD-1 / L1 small molecule antibody Fab dry powder.

[0185] Example 4

[0186] This example is used to prepare a combined antibody of broad-spectrum anti-HPV-IgY, anti-E6 / E7-IgY, anti-PD-1 / L1-IgY and their small molecule antibody Fabs.

[0187] It is formed by combining the broad-spectrum anti-HPV-IgY and its small molecule antibody Fab, anti-E6 / E7-IgY and its small molecule antibody Fab, and anti-PD-1 / L1-IgY and its small molecule antibody Fab prepared in the above Examples 1-3 respectively.

[0188] Mix the above broad-spectrum anti-HPV-IgY, broad-spectrum anti-HPV small molecule antibody Fab, anti-E6 / E7-IgY, anti-E6 / E7 small molecule antibody Fab, anti-PD-1 / L1-IgY, and anti-PD-1 / L1 small molecule antibody Fab in a ratio of (1-10):(1-10):(1-10):(1-10):(1-10):(1-10). In this example, the ratio is selected as 1:1:1:1:1:1, that is, a combined antibody of broad-spectrum anti-HPV-IgY, anti-E6 / E7-IgY, anti-PD-1 / L1-IgY and their small molecule antibody Fabs is prepared.

[0189] Example 5

[0190] In this example, an acid anhydride-modified lactoglobulin conjugated with a combined antibody of broad-spectrum anti-HPV-IgY, anti-E6 / E7-IgY, anti-PD-1 / L1-IgY and their small molecule antibody Fabs is obtained.

[0191] In the art, an acid anhydride-modified lactoglobulin is linked to a combined antibody of broad-spectrum anti-HPV-IgY, anti-E6 / E7-IgY, anti-PD-1 / L1-IgY and their small molecule antibody Fabs through a chemical linkage or genetic recombination technology. The methods that can be used include non-site-specific conjugation, site-specific conjugation by genetic engineering modification, and site-specific conjugation without relying on genetic engineering modification. At the same time, the carrier protein (linker) of the conjugate can be selected from bovine serum albumin (BSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), human serum albumin (HSA), and synthetic polylysine (PLL) and synthetic polypeptides. Among them, the conjugation by genetic recombination technology mainly uses genetic engineering technology to modify the composite antibody, so as to achieve site-specific conjugation of the acid anhydride-modified lactoglobulin and the composite antibody. The specific process will not be elaborated here.

[0192] There are also many conventional chemical linking methods in this field, such as the carbodiimide method, glutaraldehyde method, mixed anhydride method, periodate oxidation method, succinic anhydride method, carboxymethylhydroxylamine method, diazotized p-aminobenzoic acid method, sodium chloroacetate method, etc. Among them, the two main methods are the active ester method and the acid anhydride method. Heterobifunctional reagents such as SPDP can also be used as crosslinking agents for coupling, and the specific process will not be elaborated here.

[0193] During specific implementation, the linker (carrier protein) of the acyl carrier protein (MBS) activated conjugate can be used to connect the free -SH on the linker (carrier protein) of the conjugate with the side-chain -SH of Cys at the polypeptide end, improving specificity and stability. EDC, that is, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, can also be used to activate the -COOH on the linker (carrier protein) to connect the -COOH of the carrier and the -NH2 of the polypeptide. In practical applications, it is not limited to these methods.

[0194] This example illustrates with the coupling using a heterobifunctional reagent as a crosslinking agent and the glutaraldehyde method coupling as examples, and the specific operation instructions are as follows:

[0195] (1) Treat acid anhydride - modified lactoglobulin with N - succinimidyl 3 - (2 - pyridyldithio)propionate: Dissolve acid anhydride - modified lactoglobulin in a buffer solution to obtain an acid anhydride - modified lactoglobulin solution; then dissolve N - succinimidyl 3 - (2 - pyridyldithio)propionate in absolute ethanol and add it to the acid anhydride - modified lactoglobulin solution, and react at 23 - 25 °C for 30 minutes. Pass the reaction solution through a SephadexG - 25 column to remove excess SPDP and by - products, and collect and concentrate the acid anhydride - modified lactoglobulin protein solution (labeled as HRP - PDP);

[0196] (2) Treat the broad - spectrum anti - HPV - IgY and anti - PD - 1 / L1 - IgY and their small - molecule antibody Fab composite antibody with N - succinimidyl 3 - (2 - pyridyldithio)propionate: Dissolve the broad - spectrum anti - HPV - IgY and anti - PD - 1 / L1 - IgY and their small - molecule antibody Fab combined antibody in a buffer solution to obtain a combined antibody solution; then dissolve N - succinimidyl 3 - (2 - pyridyldithio)propionate in absolute ethanol and add it to the combined antibody solution, and react at 23 - 25 °C for 30 minutes. Pass the reaction solution through a SephadexG - 25 column to remove excess SPDP and by - products, and collect and concentrate the combined antibody protein solution (labeled as ANTI - PDP);

[0197] (3) Reduction of anhydrified lactoglobulin: Take the above-mentioned anhydrified lactoglobulin protein solution (labeled as HRP-PDP), add solid dithiothreitol (DTT), and react at 23-25 °C for about 25 minutes; then, pass through a Sephadex G-25 column, collect and concentrate the reduced anhydrified lactoglobulin protein solution (labeled as HRP-SH);

[0198] (4) Conjugation of anhydrified lactoglobulin and combined antibody: Mix the above-prepared reduced anhydrified lactoglobulin protein solution (labeled as HRP-SH) and the combined antibody protein solution (labeled as ANTI-PDP), react at about 4 °C for about 20 hours, and then concentrate to obtain anhydrified lactoglobulin conjugated with the combined antibody.

[0199] Similarly, this example can also be conjugated based on the glutaraldehyde method, and the specific operation is described as follows:

[0200] (1) Preparation of conjugation reaction mixture: In terms of the total amount of the entire conjugation reaction system, it includes the following components with mass contents: anhydrified lactoglobulin 10-20 wt%, broad-spectrum anti-HPV-IgY and anti-PD-1 / L1-IgY and their small molecule antibody Fab combined antibody 30-34 wt%, 0.1M pH 7.2 phosphate buffer 55-36 wt%, 0.5% glutaraldehyde 5-10 wt%;

[0201] (2) Weigh the formulated amounts of anhydrified lactoglobulin and the formulated amounts of broad-spectrum anti-HPV-IgY and anti-PD-1 / L1-IgY and their small molecule antibody Fab combined antibody respectively, place them in a powder blender and mix evenly to obtain a mixture of anhydrified lactoglobulin and combined antibody;

[0202] Add the formulated amount of 0.1M pH 7.2 phosphate buffer to the liquid reaction tank, and then add the mixture of anhydrified lactoglobulin and combined antibody, and stir well;

[0203] Continue to add the formulated amount of glutaraldehyde, adjust the temperature of the liquid reaction tank to 25 °C, stir well, and then keep the temperature of the liquid reaction tank at 25 °C and let it stand for 60 min. Obtain the conjugation reaction mixture;

[0204] (3) Separation and purification of the conjugation reaction mixture: While stirring, add saturated ferric sulfate solution to the conjugation reaction mixture at a ratio of 20-30%; then, let it stand at room temperature for 30 minutes to complete precipitation; centrifuge at 2000-3000 rpm for 10 minutes, discard the supernatant; wash the centrifuged precipitate twice with 25% saturated ferric sulfate, and then dissolve the precipitate with physiological saline; centrifuge at 3000 rpm for 15 minutes, take the supernatant, and repeat this three times; finally, dialyze with 0.15M pH7.4 phosphate buffer for 24h to obtain anhydrified lactoglobulin conjugated with the combined antibody.

[0205] Example 6

[0206] This example is used to prepare IgY against cervicitis pathogens.

[0207] To prepare the required IgY against cervicitis pathogens, it is necessary to screen representative pathogenic bacteria that cause cervicitis. According to epidemiological investigations, the main pathogens causing vaginal and cervical inflammation are Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli, Klebsiella pneumoniae, and Streptococcus agalactiae. Therefore, in this example, these five bacteria are selected as representative pathogens of cervicitis, and other vaginal and cervical inflammation pathogens can be selected for practical applications.

[0208] 1. Preparation of antigen for immunization

[0209] Cultivate pathogens: Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli, Klebsiella pneumoniae, and Streptococcus agalactiae are cultivated separately using conventional methods.

[0210] Prepare a composite antigen: First, the five cultivated pathogenic bacteria are mixed in a ratio of 1-10:1-10:1-10:1-10:1-10 (in this example, 1:1:1:1:1) and placed in a "tissue homogenizer" and thoroughly mashed and stirred evenly at 8000-12000 rpm; then, this bacterial protein mixture is added to Freund's adjuvant in a ratio of 1-10:1-10 (1:1 in this example) and homogenized at 30,000 rpm in a high-speed homogenizer to form a water-in-oil emulsion, thus obtaining the bacterial protein composite antigen.

[0211] The present invention only takes the above five bacteria as examples of representative pathogens of cervicitis, but is not limited to these five bacteria as pathogens of cervicitis.

[0212] 2. Preparation of immune eggs

[0213] In this example, egg-laying poultry with high immune response ability (such as chickens, ducks, geese, turkeys, ostriches, etc.) are selected, and the immune activation method is applied to immunize and inject with the prepared bacterial protein composite antigen. Seven days after the first injection, a second injection is given with the same dose and method. Seven days after the second injection, a third injection is given with the same dose. Starting from the 15th to 20th day after the third injection, high-immune eggs are collected.

[0214] 3. Preparation of anti-cervicitis pathogen-specific IgY

[0215] The high-immune eggs produced are respectively soaked in a 0.5% bromogeramine solution or a 0.1% KMnO4 solution or other similar disinfectants for 15 - 30 minutes for disinfection, rinsed with sterile distilled water, air-dried, the eggshells are broken in an egg beater, the egg white is filtered out with a yolk sieve, the yolk is left, 4 - 8 times distilled water is added for dilution and stirred evenly, the pH is adjusted to 5.5 - 6.0 with 1mol / L NaOH solution or 1mol / L HCl solution, left standing overnight at 4 - 6°C, the diluted solution is centrifuged at a high speed of 8000 - 12000r / min for 20 minutes, and the supernatant is taken and ultrafiltered and concentrated with an ultrafiltration machine. Then, it is purified by passing through an ion exchange column, a gel exchange column and an affinity chromatography column respectively to obtain the specific pure IgY against cervicitis pathogens.

[0216] Then, a bacterial and virus filtration device of a virus removal filtration system manufactured by Pall Ultrafine Filtration Company of the United States is used to thoroughly filter out various bacteria and viruses to ensure that the prepared IgY contains absolutely no viruses and bacteria.

[0217] In this example, after drying with a freeze dryer or medium and low temperature spray drying or fluidized bed drying and other drying methods that do not affect the antibody activity, high-purity specific pure IgY against cervicitis pathogens is prepared.

[0218] Example 7

[0219] This example is used to prepare a composition of lactoglobulin and IgY against cervicitis pathogens with a conjugated combined antibody using lipid nanoparticles as a carrier.

[0220] For the administration of conventional HPV infection drugs, due to the large number of branches and crypts in the cervical canal mucosa, pathogens hide in the crypts and mucosal spaces, and it is difficult for ordinary antibodies to enter and remove them. Therefore, the present invention selects a new generation of carriers such as lipid liquid crystalline nanoparticles (LLCN), Lipid Nanoparticle (LNP) and Solid lipid nanoparticle (SLN) as a wrapping and drug delivery system to obtain a lipid nanoparticle carrier broad-spectrum anti-HPV biological protein and IgY composition against cervicitis pathogens. Such a new generation of carriers as a wrapping and drug delivery system can increase the membrane permeability of the composition, improve the bioavailability, and have a sustained release effect; thus, significantly improve the permeability and absorption rate of the composition, can more effectively penetrate into the cervical canal crypts and mucosal spaces, produce stronger targeting, prolonged effect and permeability, and greatly improve the bioavailability and therapeutic index.

[0221] The foregoing coating process using lipid nanoparticles as a carrier in this example can adopt the conventional known coating methods in the art.

[0222] In this embodiment, one type of lipid liquid crystalline nanoparticles (LLCN) is taken as an example to illustrate the preparation method of the new carrier. The preparation of other new carriers can be implemented with reference to the conventional methods, and the specific process will not be elaborated.

[0223] Currently, the conventional preparation methods of lipid liquid crystalline nanoparticles (LLCN) in this field include mechanical stirring method, high-pressure homogenization method, heat treatment method, spontaneous emulsification method, spontaneous emulsification combined with ultrasonic technology method, low-temperature spray drying method, etc.

[0224] In the present invention, the preparation of lipid liquid crystalline nanoparticles (LLCN) by the spontaneous emulsification combined with ultrasonic technology method is taken as an example to illustrate the whole encapsulation process.

[0225] The feasible lipid liquid crystalline nanoparticle preparation system known in this field, based on its total amount, includes components with the following mass percentages. Among them, the composition and content of the excipients in the oil phase and the water phase are as follows:

[0226] Excipient components in the oil phase: fatty alcohol oils 1 - 20 wt%, non-fatty alcohol oils 0.5 - 8 wt%, emulsifiers 1 - 10 wt%;

[0227] Excipient components in the water phase: organic additives 1 - 25 wt%, inorganic additives 0.01 - 2.0 wt%, and the balance is water; among them,

[0228] The fatty alcohol oil can be selected from one or more of hexadecanol, octadecanol, docosanol, or a mixed alcohol composed of two or more of them;

[0229] The non-fatty alcohol oils can be selected from one or more of mineral oils, vegetable oils, animal oils, and synthetic oils, or a mixture composed of two or more of them;

[0230] The emulsifiers can be selected from one or more of sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, fatty alcohol polyoxyethylene ethers, polyethylene glycol fatty acid esters, ethoxylated castor oil, fatty acid monoglycerides, fatty alcohol polyglycerides, sucrose monostearate, or a mixture composed of two or more of them;

[0231] The organic additives can be selected from short-chain monohydric alcohols or polyhydric alcohols; among them, the short-chain monohydric alcohol has the molecular formula CnH2n+1OH, where n is 1 - 12, and the short-chain polyhydric alcohol is one or more of ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, propylene carbonate, and glycerol, or a mixed alcohol composed of two or more of them;

[0232] The inorganic additive can be selected as a mixture composed of one or more of sodium chloride, potassium chloride, magnesium sulfate, and sodium sulfate.

[0233] Specifically, the preparation method of the above-mentioned lipid liquid crystal nanoparticle (LLCN)-encapsulated conjugate combination antibody of lactoglobulin and anti-cervicitis pathogen IgY composition, which is feasible and known in the art, may specifically include the following steps:

[0234] (1) Heat the oil phase component composed of fatty alcohol oil and non-fatty alcohol oil and an emulsifier to 70-90°C, and stir well to obtain a uniformly dispersed oil phase component.

[0235] (2) Heat the aqueous phase component composed of an organic additive, an inorganic additive, and water to 70-90°C, stir well to form a uniformly dispersed aqueous phase component; then cool down to 60-65°C, and slowly add the conjugate combination antibody of lactoglobulin and anti-cervicitis pathogen IgY composition while stirring, and continue to stir for 60 min to obtain an aqueous solution containing the conjugate combination antibody of lactoglobulin and anti-cervicitis pathogen IgY composition.

[0236] (3) Homogenize the oil phase component at a high speed of 15000-25000 rpm, cool down to 60-65°C after homogenization, then add the aqueous solution containing the conjugate combination antibody of lactoglobulin and anti-cervicitis pathogen IgY composition thereto, continue to homogenize for 5-20 min, control the stirring speed at 300-800 rpm, and keep the temperature at 60-65°C; then, cool to room temperature to obtain the lipid liquid crystal nanoparticle (LLCN)-encapsulated conjugate combination antibody of lactoglobulin and anti-cervicitis pathogen IgY composition.

[0237] Specifically, this embodiment only takes one specific preparation process as an example for illustration, and the specific processing process is as follows:

[0238] (1) In the preparation system, based on its total amount, select the following formula: conjugate combination antibody of lactoglobulin (Example 6) and anti-cervicitis pathogen IgY composition (Example 7) 10-20 wt%, glyceryl monooleate (GMO) 30-45 wt%, poloxamer 407 (F127) 3-5 wt%, pH 5.0 phosphate buffer solution (PBS) 6-10 wt%, and the balance is distilled water.

[0239] (2) Add the formulated amount of poloxamer 407 (F127) to the formulated amount of distilled water, heat to 70-90°C, and stir well to form a uniformly dispersed aqueous phase component A.

[0240] Add the casein of the coupled combined antibody and the anti-cervicitis pathogen IgY composition in the formula amount to the phosphate buffer solution (PBS) in the formula amount, stir well to form the composition solution B;

[0241] While stirring, add the composition solution B to the aqueous phase component A to form the aqueous phase C;

[0242] Slowly add glyceryl monooleate (GMO) to the aqueous phase, stir at 50 °C at a speed of 750 r / min for 3 h to obtain the mixture D;

[0243] Place the mixture D in an ultrasonic crusher and ultrasonically crush it for 5 - 10 min (5 - 10 s each time, with an interval of 5 - 25 s between each time) to obtain the casein of the coupled combined antibody and the anti-cervicitis pathogen IgY composition with lipid nanoparticles as the carrier.

[0244] Example 8

[0245] This example provides an optoelectronic self-controlled cervical drug delivery manipulator that can accurately deliver drugs to the lesion site of the cervix and is used to spray drugs on the cervical lesion site.

[0246] As Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 6 shown, this is a specific implementation manner of the optoelectronic self-controlled cervical drug delivery manipulator provided in this example, including: a robotic arm 1, robotic fingers 3, and a light-emitting electronic eye visual component 4; the robotic arm 1 has a first chute 5 and a second chute 6, the head of the first chute 5 is provided with an opening, a push rod 7 is arranged in the first chute 5, and the push rod 7 is adapted to extrude the drug from the opening of the first chute 5, and the head of the second chute 6 is hermetically provided with a transparent electro-optical eye 10; the robotic fingers 3 are arranged at the head of the first chute 5, and a liquid outlet 9 is arranged on the outer surface of the robotic fingers 3, and the liquid outlet 9 is communicated with the first chute 5; the light-emitting electronic eye visual component 4 is inserted and arranged in the second chute 6, and the light-emitting electronic eye visual component 4 has a camera and a lighting lamp extending towards the head of the second chute 6.

[0247] During use, the robotic arm 1 is slowly inserted into the vagina. The transparent electro-optical eye 10 with a sealed head in the second chute 6 protects the camera and the lighting lamp. Through the camera and the lighting lamp of the light-emitting electro-optical eye visual component 4 extending towards the head of the second chute 6, it is possible to observe the area reached by the robotic finger 3. In addition to guiding the robotic finger 3 to find the cervical os, it is also possible to directly understand the pathological changes of the tissues around the vagina and the cervical os. After the robotic finger 3 reaches the lesion site, the push rod 7 is pressed, so that the liquid medicine is sprayed from the liquid outlet 9 on the outer surface of the robotic finger 3 onto the lesion site in the cervix. The electro-optical self-controlled cervical drug delivery manipulator provided in this embodiment ingeniously combines the three technologies of light, electricity, and machinery, and can directly deliver drugs and various preparations to the lesion site of the cervix, achieving accurate and efficient prevention and treatment effects, and solving the problem in the prior art that drugs cannot be accurately delivered to the lesion site of the cervix independently. At the same time, it can monitor the curative effect and can also be used for home care and gynecological health examinations.

[0248] Specifically, the transparent electro-optical eye can be made of transparent glass, transparent acrylic, or other transparent materials that can seal the head of the second chute 6. When the light-emitting electro-optical eye visual component 4 is inserted into the second chute 6, the camera and the lighting lamp press against the transparent electro-optical eye 10. The optimized slot design locks the distance between the camera and the lighting lamp and the transparent electro-optical eye 10 at the optimal predetermined value, preventing the transparent electro-optical eye 10 from reflecting light and affecting the clarity of the camera.

[0249] As Figure 2 、 Figure 4 shown, in the electro-optical self-controlled cervical drug delivery manipulator provided in this embodiment, a plurality of liquid outlets 9 are uniformly arranged along the axis of the robotic finger 3. During use, through the uniformly arranged plurality of liquid outlets 9, the drug can be sprayed circumferentially in the cervix, so that the drug can cover the lesion site in the cervix as much as possible. Specifically, a through liquid guiding channel is arranged inside the robotic finger 3, and the liquid guiding channel connects the liquid outlet 9 of the robotic finger 3 with the opening of the first chute 5. Additionally, as an alternative embodiment, a single liquid outlet 9 can also be provided at the head of the robotic finger 3 according to design requirements.

[0250] As Figure 1 、 Figure 3 、 Figure 4 、 Figure 6As shown in the figure, in the optoelectronic self-controlled cervical drug delivery manipulator provided in this embodiment, a syringe 2 is arranged in the first chute 5. The syringe 2 has an injection tube for accommodating drugs. A push rod 7 is inserted into the injection tube. The head of the syringe 2 is provided with an injection port 8 for extruding drugs, and the mechanical finger 3 is communicated with the injection port 8. During use, the syringe 2 with drugs is slid into the first chute 5, so that the mechanical finger 3 is communicated with the injection port 8 of the syringe 2. The push rod 7 is pushed, and the drugs are extruded from the liquid outlet 9 of the mechanical finger 3. After the drugs are used up, the syringe 2 can be taken out, and the manipulator arm 1 can be cleaned and disinfected, which is convenient for the recycling of the manipulator arm 1. In addition, as an alternative embodiment, the syringe 2 can be omitted. The first chute 5 serves as an injection chute. The mechanical finger 3 is communicated with the head opening of the first chute 5 serving as an injection chute. Drugs are poured into the first chute 5, and the drugs are extruded from the liquid outlet 9 on the mechanical finger 3 provided at the head of the first chute 5 through the push rod 7.

[0251] As Figure 1 , Figure 3 , Figure 6 , Figure 8 , Figure 10 As shown in the figure, in the optoelectronic self-controlled cervical drug delivery manipulator provided in this embodiment, it further includes a connecting sleeve 11, which is arranged in the first chute 5 of the manipulator arm 1. The head of the connecting sleeve 11 is connected to the mechanical finger 3, and the outer wall of the syringe 2 is inserted into the connecting sleeve 11. During use, the syringe 2 is inserted into the connecting sleeve 11 from the tail of the connecting sleeve 11. The head of the syringe 2 is movably connected to the tail of the mechanical finger 3, so that the injection port 8 of the syringe 2 is communicated with the mechanical finger 3 and is installed in place. By sliding the connecting sleeve 11, the mechanical finger 3 is driven to retract or extend out of the first chute 5. In addition, as an alternative embodiment, the connecting sleeve 11 can be omitted. The mechanical finger 3 can be installed at the head of the syringe 2. The assembled special injection assembly is inserted into the first chute 5, and the special injection assembly slides in the first chute 5, driving the mechanical finger 3 to retract or extend out of the first chute 5.

[0252] As Figure 1 , Figure 2 , Figure 8As shown in the figure, in the optoelectronic automatic control cervical drug delivery manipulator provided in this embodiment, the mechanical finger 3 is made of a flexible material and is bent. The syringe 2 or the connecting sleeve 11 slides in the first chute 5, driving the mechanical finger 3 to retract or extend out of the first chute 5. The flexible and bent mechanical finger 3 is squeezed by the vagina or the first chute 5, resulting in a change in the bending angle. By adjusting the position of the mechanical finger 3 in the vagina or the first chute 5, the bending angle of the mechanical finger 3 can be changed. When the mechanical finger 3 retracts into the first chute 5, it is squeezed by the first chute 5 and deforms towards a linear shape. During use, the connecting sleeve 11 of the mechanical finger 3 connected to the syringe 2 or the connecting sleeve 11 slides in the first chute 5, driving the mechanical finger 3 to retract or extend. The flexible and bent mechanical finger 3 is squeezed by the vagina or the first chute 5, causing a change in the bending shape. By adjusting the position of the mechanical finger 3 in the first chute 5, the bending angle of the mechanical finger 3 can be changed. Specifically, the bending angle of the mechanical finger 3 is between 30° and 90°.

[0253] As Figure 4 , Figure 5 , Figure 9 , Figure 10 , Figure 15 As shown in the figure, in the optoelectronic automatic control cervical drug delivery manipulator provided in this embodiment, a first card slot 12 is provided at the head of the first chute 5 of the robotic arm 1, the connecting sleeve 11, or the syringe 2. A tapered chuck 13 with reinforcing ribs is provided at the root of the mechanical finger 3. The reinforcing ribs of the tapered chuck 13 are snapped into the first card slot 12, connecting the mechanical finger 3 to the first chute 5 of the robotic arm 1, the connecting sleeve 11, or the syringe 2. During use, the tapered chuck 13 of the mechanical finger 3 is snapped into the first card slot 12 at the head of the first chute 5 of the robotic arm 1, the connecting sleeve 11, or the syringe 2, connecting the mechanical finger 3 to the first chute 5 of the robotic arm 1, the connecting sleeve 11, or the syringe 2. Through the snap connection between the reinforcing ribs of the tapered chuck 13 and the first card slot 12, the angle of the mechanical finger 3 is fixed. Additionally, as an alternative implementation, the mechanical finger 3 can also be connected to the head of the first chute 5 of the robotic arm 1, the connecting sleeve 11, or the syringe 2 by means of a threaded connection, or other means that can achieve the connection between the two.

[0254] As Figure 1 , Figure 2 , Figure 3 , Figure 8As shown, in the optoelectronic self - controlled cervical drug - delivering manipulator provided in this embodiment, the tail of the connecting sleeve 11 extends towards the outside of the robotic arm 1, and a handle 14 is provided at the tail of the connecting sleeve 11. During use, the tail of the connecting sleeve 11 extends to the outside of the robotic arm 1, and the operator slides the connecting sleeve 11 along the first chute 5 of the robotic arm 1 through the handle 14 at the tail of the connecting sleeve 11, thereby driving the mechanical finger 3 to extend out of or retract into the first chute 5 to adjust the angle of the mechanical finger 3.

[0255] As Figure 1 , Figure 6 , Figure 8 As shown, in the optoelectronic self - controlled cervical drug - delivering manipulator provided in this embodiment, a second card slot 15 is provided at the handle 14 of the connecting sleeve 11, and the shoulder 16 of the syringe 2 is snapped into the second card slot 15. During use, the shoulder 16 of the syringe 2 is snapped into the second card slot 15 at the handle 14 of the connecting sleeve 11, preventing the syringe 2 from sliding out of the connecting sleeve 11 and enabling the head of the syringe 2 to be tightly abutted against the root of the mechanical finger 3 to prevent drug leakage.

[0256] As Figure 1 , Figure 6 , Figure 8 As shown, in the optoelectronic self - controlled cervical drug - delivering manipulator provided in this embodiment, a handle 21 is provided on the shoulder 16 of the syringe 2. During use, by means of the handle 21 on the shoulder 16, the syringe 2 is rotated so that the shoulder 16 of the syringe 2 is snapped into the second card slot 15 of the handle 14, facilitating the operation.

[0257] As Figure 1 , Figure 2 , Figure 3 , Figure 7 , Figure 8 As shown, in the optoelectronic self - controlled cervical drug - delivering manipulator provided in this embodiment, a disc 19 is provided at the tail of the robotic arm 1, and a positioning groove is provided on the disc 19. Special snap - fit structures are provided on the connecting sleeve 11, the special injection assembly, and the light - emitting electronic eye visual component 4. After the snap - fit structures are aligned with the positioning groove, they are rotated and locked. During use, the disc 19 facilitates the operator to hold the robotic arm 1. After the snap - fit structures are aligned with the positioning groove and rotated and locked, it is avoided that the syringe 2, the connecting sleeve 11, the special injection assembly, and the light - emitting electronic eye visual component 4 slide out of the robotic arm 1 during use.

[0258] As Figure 1 , Figure 2 , Figure 3As shown in the figure, in the radio and television automatic control cervical drug delivery manipulator provided in this embodiment, an extending edge 20 extending outward is provided at the head of the second chute 6, and the extending edge 20 is arranged on the outer edge of the transparent photoelectric eye 10. During use, the extending edge 20 provided at the head of the second chute 6 supports the side wall tissues of the vagina and cervix, preventing the side wall tissues of the vagina and cervix from covering the transparent photoelectric eye 10, ensuring the visibility of the field of view, and being able to extend into the cervical canal to observe the lesion conditions and monitor the treatment effects.

[0259] As Figure 1 , Figure 2 , Figure 3 , Figure 7 As shown in the figure, in the photoelectric automatic control cervical drug delivery manipulator provided in this embodiment, the light-emitting electronic eye visual component 4 includes: a camera, a lighting lamp, a control box 17 and a mounting tube 18. The control box 17 is arranged at the tail of the mounting tube 18, the camera and the lighting lamp are arranged at the head of the mounting tube 18, the camera and the lighting lamp extend towards the head of the second chute 6 through the mounting tube 18, and a PCB main board, a gimbal gyroscope and a switch are arranged in the control box 17. During use, the camera and the lighting lamp of the light-emitting electronic eye visual component 4 extend towards the head of the second chute 6 through the mounting tube 18, and can observe the mechanical finger 3 part, facilitating the operator to accurately align the fingertip of the mechanical finger 3 with the cervical orifice and accurately insert it into the cervix, ensuring that the drug is delivered to the lesion part of the cervix; the control box 17 is arranged at the tail of the mounting tube 18, and the connecting wires between the camera, the lighting lamp and the control box can be arranged in the mounting tube 18, so that the control box 17 is arranged outside the robotic arm 1, facilitating the control of the light-emitting electronic eye visual component 4; through the PCB main board, gimbal gyroscope and switch arranged in the control box 17, etc., the light-emitting electronic eye visual component 4 is controlled. Specifically, the light-emitting electronic eye visual component 4 can be connected to a mobile phone, a tablet computer or other display devices by means of wires, optical fibers or wifi, Bluetooth, etc. The mounting tube 18 can be made of stainless steel pipe, plastic pipe or other materials that can realize the installation of leads.

[0260] Usage method

[0261] As Figure 1 , Figure 3 , Figure 4 , Figure 6 , Figure 8As shown in the figure, when the optoelectronic self-controlled cervical drug delivery manipulator provided in this embodiment is in use, the syringe 2 filled with the drug, the mechanical finger 3, and the connecting sleeve 11 are combined into an injection assembly. The injection assembly is inserted into the first chute 5 of the robotic arm 1, and the light-emitting electronic eye visual component 4 is inserted into the second chute 6 of the robotic arm 1. Pull the handle 14 on the connecting sleeve 11 to retract the mechanical finger 3 into the first chute 5. Slowly insert the robotic arm 1 into the vagina. Observe the position of the mechanical finger 3 through the light-emitting electronic eye visual component 4 to guide the operator to find the cervical os, and make the fingertip of the mechanical finger 3 face the cervical os exactly. Slowly insert the mechanical finger 3 into the cervical canal. After the mechanical finger 3 reaches the part where the drug needs to be sprayed, press the push rod 7 of the syringe 2 to inject the liquid medicine into the cervix from the liquid outlet 9 on the outer surface of the mechanical finger 3. Leave a part of the liquid medicine in the syringe 2. After the mechanical finger 3 is withdrawn from the cervical canal, inject the remaining liquid medicine into the vagina. Then, slowly withdraw the robotic arm 1 from the vagina, disassemble each component, and it can be reused after being disinfected with alcohol and disinfectant. The optoelectronic self-controlled cervical drug delivery manipulator provided by the drug system of the present invention can autonomously deliver drugs and various preparations directly to the cervical part, achieving accurate and efficient prevention and treatment effects, and can monitor the condition and curative effect at home.

[0262] Example 9

[0263] The optoelectronic self-controlled cervical drug delivery manipulator provided in this embodiment is generally the same in structure as that in Example 1, except that:

[0264] Such as Figure 3 、 Figure 4 、 Figure 11 、 Figure 12 、 Figure 13 、 Figure 14As shown, the syringe 2 uses a commercially available conventional vaginal applicator without a handle 21 to form another special injection component. A first card slot 12 similar to the head of the connecting sleeve 11 is provided at the head of the first chute 5 of the robotic arm 1. A conical chuck 13 is provided at the root of the robotic finger 3. The conical chuck 13 is snapped into the first card slot 12. The robotic finger 3 is fixedly or movably connected to the first card slot 12 at the head of the first chute 5 of the robotic arm 1. After the syringe 2 is filled with medicine, it is inserted into the first chute 5 of the robotic arm 1, and the syringe 2 is clamped by a circular chuck 22 with a handle 14. A disc 19 is provided at the tail of the robotic arm 1, and a positioning groove is provided on the disc 19. The circular chuck 22 is snapped into the positioning groove of the disc 19 at the tail of the robotic arm 1, and the syringe 2 is fixed by rotating the circular chuck 22 to press it tightly, preventing the syringe 2 from slipping out; the head of the syringe 2 is movably connected to the root of the robotic finger 3, so that the injection port 8 of the syringe 2 is communicated with the robotic finger 3 and is installed in place.

[0265] During use, the robotic arm 1 is inserted into the vagina. Through the camera and the illuminating lamp of the visual component 4 of the light-emitting electronic eye extending towards the head of the second chute 6, in addition to being able to observe the area reached by the robotic finger 3 and guiding the robotic finger 3 to find the cervical os, it is also possible to directly understand the pathological changes of the tissues around the vagina and the cervical os; after the robotic finger 3 reaches the lesion site, the push rod 7 of the syringe 2 is pressed, so that the liquid medicine is sprayed from the liquid outlet 9 on the outer surface of the robotic finger 3 onto the lesion site in the cervix. The optoelectronic self-controlled cervical drug delivery robotic hand provided in this embodiment combines the three technologies of light, electricity, and machinery in a clever structure, and can directly deliver drugs and various preparations to the lesion site of the cervix, achieving accurate and efficient prevention and treatment effects, and solving the problem in the prior art that drugs cannot accurately deliver drugs and various preparations to the lesion site of the cervix by themselves; at the same time, it can monitor the curative effect and can also be used for home care and gynecological health examinations.

[0266] Embodiment 10

[0267] The optoelectronic self-controlled cervical drug delivery robotic hand provided in this embodiment has substantially the same structure as that in Embodiment 1, except that:

[0268] As Figure 4 、 Figure 14 、 Figure 15 、 Figure 16As shown, the first chute 5 of the robotic arm 1 is designed as an injection chute, and a piston and the push rod 7 are installed in the first chute 5; a first clamping groove 12 is provided at the opening of the first chute 5 of the robotic arm 1, and the conical chuck 13 of the robotic finger 3 is snapped into the first clamping groove 12, so that the liquid outlet 9 of the robotic finger 3 communicates with the first chute 5. The first chute 5 is pre-filled with drugs or various preparations, and the opening of the first chute 5 is sealed with a special plug; during use, the plug is removed, and the robotic finger 3 is installed at the opening of the first chute 5 through the first clamping groove 12 provided at the opening of the first chute 5 of the robotic arm 1. A disc 19 is provided at the tail of the robotic arm 1, a positioning groove is provided on the disc 19, and a special snap structure is provided on the light-emitting electronic eye visual component 4. After the snap structure is aligned with the positioning groove, it is rotated and locked to prevent the light-emitting electronic eye visual component 4 from slipping out. The robotic arm 1 is inserted into the vagina. Through the camera and the illuminating lamp extending towards the head of the second chute 6 on the light-emitting electronic eye visual component 4, in addition to being able to observe the area reached by the robotic finger 3 and guiding the robotic finger 3 to find the cervical os, it is also possible to directly understand the pathological changes of the vaginal and cervical os peripheral tissues. After the robotic finger 3 reaches the lesion site, the push rod 7 is pressed, so that the liquid medicine is sprayed from the liquid outlet 9 on the outer surface of the robotic finger 3 onto the lesion site in the cervix. The optoelectronic self-controlled cervical drug delivery manipulator provided in this embodiment ingeniously combines the three technologies of light, electricity, and machinery, and can directly deliver drugs and various preparations to the lesion site of the cervix, achieving accurate and efficient prevention and treatment effects, and solving the problem in the prior art that drugs and various preparations cannot be accurately delivered to the lesion site of the cervix independently; at the same time, it can monitor the curative effect and can also be used for home care and gynecological health examinations.

[0269] The optoelectronic self-controlled cervical drug delivery manipulator of the present invention effectively solves the problem of cervical drug administration. The nano-particle preparation of the conjugate combination antibody of lactoglobulin and anti-cervicitis pathogen IgY composition can be directly made into stable preparations of various dosage forms, or can be added with excipients to make stable preparations of various dosage forms; the stable preparations are at least one of gel, dressing, spray, gynecological lotion and men's lotion, hand sanitizer, powder, tablet, toothpaste, oral paste, mouthwash, buccal tablet, oral liquid, oral preparation, capsule, etc., but the stable preparations include but are not limited to the above preparations; the excipients are one or more of excipients, fillers, solvents, pastes, solubilizers, surfactants and capsule excipients. The stable preparations of various dosage forms are added to an injection tube and then placed into the optoelectronic self-controlled cervical drug delivery manipulator, and the various preparations are sprayed onto the lesion site of the cervix by means of the optoelectronic self-controlled cervical drug delivery manipulator.

[0270] Experimental Example

[0271] The following experimental example part of the present invention will be described in detail through specific experimental test examples and embodiments.

[0272] Experimental Example 1

[0273] This experimental example is used to detect the antibody binding titer of the broad-spectrum anti-HPV-IgY antibody (Example 1) against 15 high-risk human papillomavirus (HPV) types.

[0274] HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, and HPV82 were respectively selected as detection antigens, and the antibody titer of the prepared broad-spectrum anti-HPV-IgY was detected by the "ELISA" method (enzyme-linked immunosorbent assay). The results are shown in Table 1 below.

[0275] Table 1 Detection results of binding titer

[0276]

[0277] Note: The concentration of the broad-spectrum anti-HPV-IgY antibody solution in the test sample is 1 mg / ml.

[0278] It can be seen from the above detection results that the broad-spectrum anti-HPV-IgY antibody prepared by the present invention has a high antibody binding titer against 15 high-risk human papillomavirus (HPV) antigens.

[0279] Experimental Example 2

[0280] This example is used to detect the antibody binding titer of the broad-spectrum anti-HPV-Fab small molecule antibody against 15 high-risk human papillomavirus (HPV) types.

[0281] HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, and HPV82 were respectively selected as detection antigens, and the antibody titer of the prepared broad-spectrum anti-HPV-Fab small molecule antibody (Example 1) was detected by the "ELISA" method (enzyme-linked immunosorbent assay). The results are shown in Table 2 below.

[0282] Table 2 Detection results of binding titer

[0283]

[0284]

[0285] Note: The concentration of the broad-spectrum anti-HPV-Fab small molecule antibody solution in the test sample is 1 mg / ml.

[0286] From the above test results, it can be seen that the prepared broad-spectrum anti-HPV-Fab small molecule antibody has a high antibody binding titer against 15 high-risk human papillomavirus (HPV) antigens.

[0287] Experimental Example III

[0288] This experimental example is used to detect the antibody binding titers of the broad-spectrum anti-HPV-E6 / E7-IgY and the anti-HPV-E6 / E7 small molecule antibody Fab against 15 kinds of HPV-E6 / E7 oncogene proteins.

[0289] HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV31-E6, HPV31-E7, HPV33-E6, HPV33-E7, HPV35-E6, HPV35-E7, HPV39-E6, HPV39-E7, HPV45-E6, HPV45-E7, HPV51-E6, HPV51-E7, HPV52-E6, HPV52-E7, HPV56-E6, HPV56-E7, HPV58-E6, HPV58-E7, HPV59-E6, HPV59-E7, HPV68-E6, HPV68-E7, HPV73-E6, HPV73-E7, HPV82-E6, and HPV82-E7 were respectively selected as the detection antigens, and the antibody titers of the prepared broad-spectrum anti-HPV-E6 / E7-IgY were detected by the "ELISA" method (enzyme-linked immunosorbent assay). The results are shown in Table 3 below.

[0290] Table 3 Detection results of binding titers

[0291]

[0292]

[0293] Note: The concentration of the broad-spectrum anti-HPV-E6 / E7-IgY antibody solution in the test sample is 1 mg / ml.

[0294] From the above test results, it can be seen that the broad-spectrum anti-HPV-E6 / E7-IgY antibody prepared by the present invention has a high antibody binding titer against 15 high-risk HPV-E6 / E7 oncogene proteins, proving that this broad-spectrum anti-HPV-E6 / E7-IgY antibody has ideal broad-spectrum properties.

[0295] Experimental Example IV

[0296] This experimental example is used to detect the antibody binding titer of IgY against cervical inflammation pathogens to representative cervical inflammation pathogens.

[0297] Using Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli, Klebsiella pneumoniae, and Streptococcus agalactiae as detection antigens respectively, the antibody binding titer of the prepared IgY against cervical inflammation pathogens (Example 6) was detected by the "ELISA" method, and the results are shown in Table 4 below.

[0298] Table 4 Detection results of binding titer

[0299]

[0300]

[0301] Note: The concentration of the IgY solution against cervical inflammation pathogens in the test sample is 1 mg / ml.

[0302] It can be seen from the above detection results that the prepared IgY against cervical inflammation pathogens has a high antibody binding titer to 5 representative cervical inflammation pathogen antigens.

[0303] Experimental Example Five

[0304] In this experimental example, the optoelectronic automatic cervical drug delivery manipulator described in Example 9 was used to spray the nanometer particle gel preparation of the conjugate combination antibody of lactoglobulin and IgY against cervical inflammation pathogens prepared in Example 7 into the cervix of HPV-infected positive patients to observe the curative effect.

[0305] General information

[0306] Selection criteria: (1) Aged between 25 and 50 years old, with a history of sexual life; (2) TCT (-), HPV (-) in cervical cancer screening within 3 years; (3) TCT (-) in this cervical cancer screening, and any one or more of the 15 high-risk HPV types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82) are (+).

[0307] Exclusion criteria: (1) History of cervical cancer and precancerous lesions in the past; (2) HPV (+) in the past; (3) Other HPV types (+) other than the 15 high-risk HPV types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82) in this cervical cancer screening; (4) History of hysterectomy; (5) Currently pregnant; (6) Patients without follow-up conditions.

[0308] A total of 160 women meeting the above criteria were selected as the research subjects in this study. The research subjects were randomly divided into group A (treatment group) with 81 cases and group B (control group) with 79 cases. There were no significant differences in general data such as age and disease course between the two groups of patients.

[0309] Before treatment, the above FQ-PCR method was used to detect and count the positive rates of the treatment group and the control group respectively.

[0310] Treatment method

[0311] The patients in group A (treatment group) were treated with a nanoparticle gel preparation of lactoglobulin conjugated with a combination antibody and an IgY composition against cervicitis pathogens for 30 days through a photoelectrically controlled cervical drug delivery manipulator. After rinsing the vagina before going to bed, the nanoparticle gel preparation of lactoglobulin conjugated with a combination antibody and an IgY composition against cervicitis pathogens was loaded into the injection tube of the photoelectrically controlled cervical drug delivery manipulator according to the instructions, and then the "miniature light-emitting electronic eye assembly" was inserted. According to the display on the mobile phone screen of the light-emitting electronic eye visual electronic eye, the fingertip of the flexible cervical drug delivery mechanical finger was aligned with the cervical os; then, the flexible cervical drug delivery mechanical finger was slowly inserted into the cervical canal, and then the nanoparticle gel was injected into the cervical canal to complete cervical drug administration.

[0312] The patients in group B (control group) were treated with recombinant human interferon α2a suppository for 30 days. After rinsing the vagina before going to bed, 1 suppository of recombinant human interferon α2a was injected into the posterior fornix of the vagina or the vaginal stump, 1 suppository was used every night, and 30 days was 1 course of treatment.

[0313] It should be noted that sitz baths and sexual intercourse are prohibited during the medication period. After 30 days, the above FQ-PCR method was used to detect and count the positive rates of the treatment group and the control group respectively.

[0314] Instruments and reagents

[0315] Hema3200 type polymerase chain reaction (PCR) amplifier, DA800 type fully automatic nucleic acid molecule hybridization instrument, HPV gene typing detection kit.

[0316] Detection method

[0317] Specimen collection: Expose the cervix with a speculum, wipe the cervical secretions with a cotton swab, then place a special cervical brush at the cervical os and rotate the cervical brush 4 - 5 times to obtain a sufficient amount of cervical epithelial cell specimens; take out the cervical brush, put it into a special tube containing cell preservation solution, break the brush handle along the crease of the brush handle, tighten the tube cap and send it for inspection in time.

[0318] Detection method: Extract DNA by conventional method; after adding DNA and reagents according to the requirements of the kit instructions, perform cycling 40 times at 93°C for 3 min, 93°C for 40 s, 55°C for 40 s, and 72°C for 40 s; amplify at 72°C for 7 min; after the amplification product is denatured at 98°C for 8 min, immediately place it in ice water; perform hybridization on an automatic nucleic acid molecule hybridization instrument. The result interpretation refers to the kit instructions. When the HPV-DNA of any type exceeds the threshold, the test result is positive. Patients with the HPV-DNA of one or more types of HPV exceeding the threshold are all judged as positive patients, and the positive ratio is calculated accordingly.

[0319] Statistical method

[0320] Use SPSS 17.0 statistical software to perform statistical analysis on the collected data. For count data, use the t-test, and for measurement data, use the X2 test. When P < 0.05, it indicates that the difference is statistically significant.

[0321] Experimental results

[0322] Use the above method to detect group A (treatment group) and group B (control group) respectively, and calculate the positive rates of group A (treatment group) and group B (control group) respectively. The detailed situation is shown in Table 5 below.

[0323] Table 5 Clinical trial results

[0324]

[0325] The above patients were reexamined 90 days after the end of a treatment course (no sexual life during this period). In the treatment group, the number of patients with HPV-DNA exceeding the threshold decreased from 81 cases to 7 cases, and the negative conversion rate was 91.4%; in the control group, the number of patients with HPV-DNA exceeding the threshold decreased from 79 cases to 51 cases, and the negative conversion rate was 35.4%. P < 0.05, showing a significant difference.

[0326] This experimental example shows that the nanometer particles of the combination antibody-coupled lactoglobulin and anti-cervicitis pathogen IgY composition of the present invention, when administered in combination with the optoelectronic automatic cervical drug delivery manipulator, have a good therapeutic effect on patients infected with HPV.

[0327] Experimental example six

[0328] In this experimental example, the optoelectronic automatic cervical drug delivery manipulator described in Example 10 was used to spray the nanometer particle gel preparation of the combination antibody-coupled lactoglobulin and anti-cervicitis pathogen IgY composition prepared in Example 7 into the cervix of patients with cervicitis to observe the curative effect.

[0329] General information

[0330] 120 out - patient cases diagnosed with mild - to - moderate cervical erosion were randomly divided into a control group of 58 cases and a treatment group of 62 cases. In the control group of 58 cases, the age ranged from 18 to 45 years old, with an average age of (35.1 ± 1.7) years; in the treatment group of 62 cases, the age ranged from 19 to 47 years old, with an average age of (35.7 ± 1.3) years. There were no statistically significant differences in age, disease course, and disease condition between the two groups (P > 0.05), and they were comparable.

[0331] Diagnostic criteria

[0332] According to the erosion area, it was divided into mild, moderate, and severe degrees. When the erosion area < 1 / 3 of the total cervical area, it was mild (I°); when it accounted for 1 / 3 - 1 / 2, it was moderate (II°); when it > 1 / 2, it was severe (III°).

[0333] According to the degree of inflammation and the growth rate of columnar epithelium, cervical erosion can be manifested in three types: (1) Simplex type: In the initial stage of inflammation, the erosion surface is covered by a single - layer columnar epithelium, with a flat surface and a smooth appearance; (2) Granular type: Due to excessive hyperplasia of cervical glandular epithelium and stromal hyperplasia, the erosion surface is uneven and appears granular; (3) Papillary type: The hyperplasia of glandular epithelium and stroma is significant, and the surface unevenness is more obvious, forming papillary protrusions.

[0334] Treatment method

[0335] Treatment group: The nano - particle gel preparation of the lactoglobulin and anti - cervicitis pathogen IgY composition conjugated with the antibody combination in Example 7 of the present invention was used. After cleaning the vulva before going to bed, according to the instructions of the photoelectric self - controlled cervical drug - delivery manipulator operation method, the nano - particle gel was injected into the cervix through the photoelectric self - controlled cervical drug - delivery manipulator. One 3 - g tube was used every night, and 15 days was one course of treatment.

[0336] Control group: Recombinant human interferon α2a suppository was used for treatment. After flushing the vagina before going to bed, one suppository of recombinant human interferon α2a was pushed into the posterior fornix of the vagina or the vaginal stump. Once a night, one suppository each time, and 15 days was one course of treatment.

[0337] It should be noted that during the treatment of the two groups, sexual intercourse and sitz baths were prohibited, and the drugs were stopped during menstruation.

[0338] Efficacy judgment criteria

[0339] Cured: The cervix is smooth, the erosion surface disappears, and the clinical symptoms completely disappear;

[0340] Markedly effective: The clinical symptoms are significantly reduced, the area of cervical erosion is reduced, and the depth becomes shallower;

[0341] Ineffective: The clinical symptoms are not reduced, and there are no obvious changes in the cervical erosion surface and depth.

[0342] Efficiency = cure rate + marked improvement rate.

[0343] Statistical processing

[0344] The SPSS 11.0 statistical software was used to analyze the data. The t-test was used for comparison of the clinical data between the two groups, and the r-test was used for comparison of the efficiency and cure rate of the drug treatment between the two groups.

[0345] Experimental results

[0346] The total efficiency rate of the treatment group was 90.4%, and that of the control group was 53.5%. The curative effect of the treatment group was significantly better than that of the control group, with a significant difference (P < 0.05), as shown in Table 6 below.

[0347] Table 6 Comparison of the clinical effects of the two groups of patients (n, %)

[0348] Group n Cured Markedly effective Improved Invalid Cure rate (%) Total effective rate (%) Treatment group 62 56 5 1 0 90.3 90.4 Control group 58 31 3 8 16 53.4 53.5

[0349] This experimental example shows that the use of the nanoparticles of the lactoglobulin and anti-cervicitis pathogen IgY composition conjugated with the antibody combination of the present invention, combined with the optoelectronic automatic cervical drug delivery manipulator for drug delivery, has a good therapeutic effect on cervicitis patients.

[0350] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A composition for anti-HPV infection, characterized in that, The composition comprises lactoglobulin conjugated with a combined antibody and IgY against cervicitis pathogens; Preferably, the mass ratio of the lactoglobulin conjugated with the combined antibody to IgY against cervicitis pathogens is (1 - 10):(1 - 10).

2. The composition for anti-HPV infection according to claim 1, wherein In the lactoglobulin conjugated with the combined antibody, the combined antibody comprises a mixture of broad-spectrum anti-HPV-IgY and its small molecule antibody Fab, composite anti-E6 / E7-IgY and its small molecule antibody Fab, and anti-PD-1 / L1-IgY and its small molecule antibody Fab; Preferably, the mass ratio of the broad-spectrum anti-HPV-IgY, the composite anti-E6 / E7-IgY, and the anti-PD-1 / L1-IgY is (1 - 10):(1 - 10):(1 - 10); Preferably, the mass ratio of the broad-spectrum anti-HPV-IgY, the small molecule antibody Fab of the broad-spectrum anti-HPV-IgY, the composite anti-E6 / E7-IgY, the small molecule antibody Fab of the composite anti-E6 / E7-IgY, the anti-PD-1 / L1-IgY, and the small molecule antibody Fab of the anti-PD-1 / L1-IgY is (1 - 10):(1 - 10):(1 - 10):(1 - 10):(1 - 10):(1 - 10).

3. The composition for anti-HPV infection according to claim 2, wherein The preparation method of the lactoglobulin conjugated with the combined antibody comprises: (a) Preparing the required antigens respectively according to the selected antibody types; (b) Preparing the corresponding immune eggs respectively by using the prepared antigens; (c) Preparing the required types of antibody-IgY and its small molecule antibody Fab respectively, and obtaining the required combined antibody; (d) Conjugating the combined antibody with acid anhydride-modified lactoglobulin to obtain the product.

4. The composition for anti-HPV infection according to any one of claims 1-3, characterized in that The pathogens of the IgY against cervicitis pathogens include the main pathogen types causing vaginitis; Preferably, the pathogens include a mixture of Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli, Klebsiella pneumoniae, and Streptococcus agalactiae.

5. The composition for anti-HPV infection according to claim 3 or 4, wherein The preparation method of the IgY against cervicitis pathogens comprises: (a) Culturing the selected types of pathogens and preparing the corresponding composite antigen; (b) Preparing the immune egg by using the composite antigen; (c) Preparing the IgY against cervicitis pathogens by using the immune egg to obtain the product.

6. A nanoparticle against HPV infection, characterized in that, The nanoparticle comprises the anti-HPV infection composition according to any one of claims 1 - 5.

7. The nanoparticle for anti-HPV infection according to claim 6, characterized in that, The nanoparticle comprises the anti-HPV infection composition encapsulated by a liposome as a carrier; Preferably, the liposome comprises at least one of lipid liquid crystal nanoparticles, lipid nanoparticles, or solid lipid nanoparticles.

8. An external preparation for anti-HPV infection, characterized in that, Comprising the anti-HPV infection composition according to any one of claims 1 - 5 and / or the anti-HPV infection nanoparticle according to claim 6 or 7, and acceptable excipients.

9. The external preparation according to claim 8, characterized in that, The preparation comprises a gel, a dressing, a spray, a gynecological lotion, a men's lotion, a hand sanitizer, a powder, a tablet, a toothpaste, an oral paste, a mouthwash, a buccal tablet, an oral liquid, an oral preparation, or a capsule.

10. A drug system for anti-HPV infection, characterized in that, Comprising the anti-HPV infection topical preparation according to claim 8, and an optoelectronic self-controlled cervical drug delivery manipulator.

11. The pharmaceutical system according to claim 10, wherein The optoelectronic self-controlled cervical drug delivery manipulator is characterized in that it comprises: The robotic arm (1) has a first chute (5) and a second chute (6). The head of the first chute (5) is provided with an opening. A push rod (7) is arranged in the first chute (5). The push rod (7) is adapted to extrude the drug from the opening of the first chute (5). The head of the second chute (6) is hermetically provided with a transparent photoelectric eye (10). The robotic finger (3) is arranged at the head of the first chute (5). The outer surface of the robotic finger (3) is provided with a liquid outlet (9). The liquid outlet (9) is communicated with the first chute (5). The light-emitting photoelectric eye visual component (4) is inserted and arranged in the second chute (6). The light-emitting photoelectric eye visual component (4) has a camera and a lighting lamp extending towards the head of the second chute (6).

12. Use of the anti-HPV infection composition according to any one of claims 1-5 and / or the anti-HPV infection nanoparticle according to claim 6 or 7 for preparing a drug for preventing and treating HPV infection, cervical infectious diseases or cervical cancer; or, Use of the anti-HPV infection composition according to any one of claims 1-5 and / or the anti-HPV infection nanoparticle according to claim 6 or 7 for preparing a non-diagnostic detection reagent or detection product; or, Use of the anti-HPV infection composition according to any one of claims 1-5 and / or the anti-HPV infection nanoparticle according to claim 6 or 7 for preparing a medical device for anti-HPV infection.

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