Recombinant bioactive protein for preventing or treating human papilloma virus infection as well as preparation method and application thereof

Abstract

The invention relates to a recombinant bioactive protein for preventing or treating human papilloma virus infection as well as a preparation method and application thereof, and belongs to the technical field of medicines. The recombinant bioactive protein disclosed by the invention is anhydridized recombinant lactoferrin, anhydridized recombinant lactoglobulin or anhydridized recombinant histone; the recombinant bioactive protein is obtained by carrying out anhydridation modification on recombinant lactoferrin, recombinant lactoglobulin or recombinant histone by using 3-hydroxyphthalic anhydride or a derivative thereof, 4-hydroxyphthalic anhydride or a derivative thereof, succinic anhydride or maleic anhydride. The bioactive protein disclosed by the invention has the advantages of preventing HPV virus from invading cells, preventing virus from expanding infection, resisting bacteria and oxidation, being safe, stable, convenient to store and transport, low in cost and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a recombinant bioactive protein for preventing or treating human papillomavirus infection, a preparation method thereof and an application thereof, and belongs to the technical field of medicine. BACKGROUND

[0002] Human papillomavirus (HPV) is one of the few viruses that are clearly associated with human cancer, and persistent infection is an important risk factor for the occurrence of cervical cancer. According to statistics, more than 99% of all cervical cancer patients can be detected with high-risk HPV DNA. More than 50% of sexually active women have had HPV infection in their lifetime, and persistent high-risk HPV infection increases the risk of cervical cancer by 100-300 times. Therefore, early intervention and clearance of HPV infection is very important for the prevention of cervical lesions CIN. At present, there are many treatments for HPV infection, among which local medication is one of the commonly used methods, and there are many types of drugs, but the clinical cure rate is not ideal.

[0003] At present, there are commercialized bivalent (HPV-16, 18 type) or quadrivalent HPV vaccines (HPV-6, 11, 16 and 18 types) that can be used to prevent infection with the same type of virus. However, the vaccine is too expensive, and the specific vaccine does not have cross-immunity to other types of HPV. At present, there are few drugs and biological agents on the market for preventing and treating human papillomavirus infection, and new drugs and biological agents still need to be developed. The use of gel dressings can increase the residence time of drugs at the site of action and improve the efficacy of drugs.

[0004] In Chinese patent 202110238692.3, a bioactive protein for preventing and treating human papillomavirus infection, a preparation method thereof and an application thereof are provided, which discloses a charge-modified lactoferrin combined with carbomer 934, including 0.01-0.1% of anhydride-modified lactoferrin freeze-dried powder, 1-2% of carbomer 934, 0.5%-2% of glycerol and the balance of purified water. The combined drug can be a liquid preparation, a paste, a gel or a suppository. The lactoferrin used in the patent is modified by 4-hydroxy-phthalic anhydride on bovine lactoferrin. Since bovine lactoferrin is derived from animals, its safety is lower than that of recombinant lactoferrin, its application range is smaller than that of recombinant lactoferrin, and it may affect the effect of anti-HPV virus. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provide a recombinant bioactive protein for preventing or treating human papillomavirus infection, a preparation method thereof and an application thereof.

[0006] The inventive concept of the present application is as follows: the recombinant lactoferrin is modified to obtain anhydride-modified recombinant lactoferrin, which has more negative charges on the surface, and is combined with the positive charge region of the C-terminal L2 region of the L1 region of HPV capsid protein, thereby blocking the process of virus invasion into host cells, and achieving the purpose of blocking HPV infection. Through a large amount of research work, the inventors unexpectedly found that 4-hydroxyphthalic anhydride has a higher modification rate for recombinant lactoferrin, a higher amount of negative charges after modification, and a better effect, and the obtained anhydride-modified recombinant lactoferrin has a better anti-HPV virus effect.

[0007] The technical solution of the present application to solve the technical problems is as follows:

[0008] In the first aspect of the present application, an anhydride-modified recombinant lactoferrin, an anhydride-modified recombinant lactoglobulin or an anhydride-modified recombinant histatin is provided for preventing or treating human papilloma virus infection.

[0009] Preferably, the recombinant bioactive protein is anhydride-modified recombinant lactoferrin modified by 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride.

[0010] Also preferably, the recombinant bioactive protein is anhydride-modified recombinant lactoglobulin modified by 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride.

[0011] In the second aspect of the present application, a preparation method of the recombinant bioactive protein of the first aspect is provided.

[0012] When the recombinant bioactive protein is anhydride-modified recombinant lactoferrin modified by 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride, the preparation method comprises the following steps: dissolving 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride in dimethyl sulfoxide at a temperature of 25-50℃ to obtain a saturated 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride solution; dissolving recombinant lactoferrin in 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30-80mg / ml; adding the above-mentioned 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride solution to the protein solution, stirring to mix, and the final concentration of 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride in the reaction system is 30-80mM, standing for 2 hours to obtain anhydride-modified recombinant lactoferrin; after sterilization by filtration with a 0.45um filter membrane, performing ultrafiltration displacement purification (the ultrafiltration membrane used has a molecular weight cut-off of 10KD) or ion exchange purification with a pH 7.2 phosphate buffer; again sterilizing by filtration with a 0.45um microporous filter membrane, adding a protective agent, and performing freeze-drying with a freeze-drying machine to obtain anhydride-modified recombinant lactoferrin freeze-dried powder.

[0013] Preferably, the protein lyophilization step comprises the following stages: pre-freezing stage: the temperature of the shelf is lowered to -80℃, and the holding time is 6h; primary sublimation stage: the temperature of the shelf is raised to -25℃, and the holding time is 12h; secondary drying stage: the temperature of the shelf is raised to 20℃, and the holding time is 8h. The lyophilization protective agent is mannitol, sucrose, glucose, trehalose or fructose.

[0014] In the third aspect of the present application, the use of the recombinant bioactive protein as described in the first aspect in the preparation of a medicament for preventing or treating human papillomavirus infection is provided.

[0015] In the fourth aspect of the present application, a pharmaceutical preparation containing the recombinant bioactive protein as described in the first aspect is provided, wherein the preparation contains a pharmaceutically effective dose of the recombinant bioactive protein and pharmaceutically acceptable auxiliary ingredients.

[0016] Preferably, the preparation can be a liquid preparation (such as a dressing), a paste, a gel or a suppository. For the preparation of a solid preparation for external administration, a solid carrier known in the art such as carbomer, carrageenan, malt dextrin, hydroxypropyl methylcellulose or gelatin can be used. In addition, any other carrier commonly used for the purpose of preservation can also be used as long as it is compatible with the active ingredient or the used ingredient. For the preparation of a liquid dosage form for external administration, water, ethanol, glycerol or propylene glycol and other liquid carriers known in the art can be used.

[0017] Preferably, the preparation is a dressing, and the recombinant bioactive protein in the preparation is 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride modified anhydrous recombinant lactoferrin. The components of the dressing and their weight percentages are as follows: anhydrous recombinant lactoferrin freeze-dried powder 0.001-0.1%, carbomer 934 1-2%, glycerol 0.5%-2%, and the balance is purified water.

[0018] The preparation method of the dressing comprises the following steps:

[0019] 1) Put the carbomer into purified water and stir to dissolve, and then stand for dissolution.

[0020] 2) Dissolve the anhydrous recombinant lactoferrin in an appropriate amount of purified water, then add it to the solution of step 1), and then add glycerol, stir for 20-60 minutes to mix evenly, and then add purified water and stir for 30 minutes to obtain the dressing.

[0021] The present application has the following technical effects:

[0022] 1. The recombinant bioactive protein of the present application has the advantages of blocking HPV virus invasion of cells, preventing virus from expanding infection, safety, stability, easy storage and transportation, and low cost.

[0023] 2. Compared with the existing technology, namely, application number 202110238692.3, patent title "A bioactive protein for the prevention and treatment of human papillomavirus infection, its preparation method and its application", the technical solution of the present invention has the following advantages:

[0024] 1) In this invention, the concentration of recombinant lactoferrin and the preparation process were optimized to ensure that the negative charge of the modified recombinant lactoferrin is maximized.

[0025] 2) This invention unexpectedly discovered that when recombinant lactoferrin is modified with 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride, the recombinant lactoferrin has less steric hindrance, higher activity, higher modification rate and better modification effect compared with the original bovine lactoferrin, and is also more widely available and cheaper.

[0026] 3) This invention employs lyophilization to preserve anhydride-modified recombinant proteins. Since anhydride-modified recombinant protein solutions are easily degraded at room temperature or 4°C, they are difficult to preserve long-term. This invention uses lyophilization to preserve anhydride-modified recombinant proteins, solving the problem of long-term preservation of recombinant protein activity.

[0027] 4) This invention employs ultrafiltration replacement purification or ion exchange purification methods to purify proteins. Ultrafiltration replacement of anhydride-modified recombinant lactoferrin results in faster ultrafiltration speed, shorter processing time, higher concentration, better ultrafiltration effect, and higher yield compared to anhydride-modified bovine lactoferrin. This method, using ultrafiltration replacement purification or ion exchange purification, is more conducive to industrial production.

[0028] In summary, the protein preparation process of this invention is simple, stable, effective, safe, reliable, and easy to promote. The dressing contains carbomer, which can adsorb and encapsulate inactivated HPV, expel it from the body, accelerate the rapid repair and regeneration of damaged and eroded tissues, and promote healing. It is suitable for isolating and blocking HPV infection, promoting the healing of damaged and eroded tissues, and has preventive and therapeutic effects on cervical cancer. Attached Figure Description

[0029] Figure 1 The results of the modified lactoferrin potential detection were obtained using 4-hydroxyphthalic anhydride (existing patent 202110238692.3).

[0030] Figure 2 The results of potential detection for modified recombinant lactoferrin obtained using 4-hydroxyphthalic anhydride are shown.

[0031] Figure 3The results are for the detection of Anti-HPV 16E7 antibody. Among them, 1 represents normal HeLa cells; 2 represents total cell protein in cells after 24 hours of a 1 mg / ml sample; 3 represents total cell protein in cells after 48 hours of a 1 mg / ml sample; 4 represents total cell protein in cells after 24 hours of a 0.5 mg / ml sample; 5 represents total cell protein in cells after 48 hours of a 0.5 mg / ml sample; 6 represents total cell protein in cells after 24 hours of a 0.25 mg / ml sample; and 7 represents total cell protein in cells after 48 hours of a 0.25 mg / ml sample.

[0032] Figure 4 The results are for the detection of Anti-HPV 18E7 antibody. Among them, 1 represents normal HeLa cells; 2 represents total cell protein in cells after 24 hours of a 1 mg / ml sample; 3 represents total cell protein in cells after 48 hours of a 1 mg / ml sample; 4 represents total cell protein in cells after 24 hours of a 0.5 mg / ml sample; 5 represents total cell protein in cells after 48 hours of a 0.5 mg / ml sample; 6 represents total cell protein in cells after 24 hours of a 0.25 mg / ml sample; and 7 represents total cell protein in cells after 48 hours of a 0.25 mg / ml sample. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0034] The sources of the materials involved in this invention are explained as follows:

[0035] The recombinant lactoferrin described in this invention is plant-derived recombinant human lactoferrin OsrhLF, purchased from Wuhan Heyuan Biotechnology Co., Ltd., catalog number: HYC004M01, molecular weight 80kD, CAS number: 936541-36-5.

[0036] Example 1: Preparation of 4-hydroxyphthalic anhydride-modified anhydride-modified recombinant lactoferrin

[0037] At 25°C, 4-hydroxyphthalic anhydride was dissolved in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; recombinant lactoferrin was dissolved in 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30 mg / ml; the above 4-hydroxyphthalic anhydride solution was added to the protein solution, stirred and mixed, the final concentration of 4-hydroxyphthalic anhydride in the reaction system was 60 mM, and the reaction was allowed to stand for 2 hours to obtain anhydride-modified recombinant lactoferrin; after sterilization by filtration through a 0.45 μM filter membrane, ultrafiltration replacement purification was performed using pH 7.2 phosphate buffer, with the ultrafiltration membrane having a molecular weight cutoff of 10 KD; after sterilization by filtration through a 0.45 μM microporous filter membrane, mannitol was added as a protective agent, and the mixture was freeze-dried using a freeze dryer, and the lyophilized powder was collected to obtain anhydride-modified recombinant lactoferrin lyophilized powder.

[0038] The protein freeze-drying process includes the following stages: Pre-freezing stage: the temperature of the separator is lowered to -80℃ and held for 6 hours; First sublimation stage: the temperature of the separator is raised to -25℃ and held for 12 hours; Second drying stage: the temperature of the separator is raised to 20℃ and held for 8 hours.

[0039] Example 2: Preparation of 4-hydroxyphthalic anhydride-modified anhydride-modified recombinant lactoferrin

[0040] At 35°C, 4-hydroxyphthalic anhydride was dissolved in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; recombinant lactoferrin was dissolved in 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 50 mg / ml; the above 4-hydroxyphthalic anhydride solution was added to the protein solution, stirred and mixed, with a final concentration of 80 mM for 4-hydroxyphthalic anhydride in the reaction system, and allowed to stand for 2 hours to obtain anhydride-modified recombinant lactoferrin; after sterilization by filtration through a 0.45 μM filter membrane, ultrafiltration was performed using pH 7.2 phosphate buffer for replacement purification, with the ultrafiltration membrane having a molecular weight cutoff of 10 KD; after sterilization by filtration through a 0.45 μM microporous filter membrane, trehalose was added as a protective agent, and the mixture was freeze-dried using a freeze dryer. The lyophilized powder was collected to obtain the anhydride-modified recombinant lactoferrin lyophilized powder.

[0041] The protein freeze-drying procedure is the same as in Example 1.

[0042] Example 3: Preparation of 4-hydroxyphthalic anhydride-modified anhydride-modified recombinant lactoferrin

[0043] At 50°C, 4-hydroxyphthalic anhydride was dissolved in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; recombinant lactoferrin was dissolved in 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 80 mg / ml; the above 4-hydroxyphthalic anhydride solution was added to the protein solution, stirred and mixed, the final concentration of 4-hydroxyphthalic anhydride in the reaction system was 80 mM, and the reaction was allowed to stand for 2 hours to obtain anhydride-modified recombinant lactoferrin; after sterilization by filtration through a 0.45 μM filter membrane, ultrafiltration was performed using pH 7.2 phosphate buffer for replacement purification, the molecular weight cutoff of the ultrafiltration membrane was 10 KD; after sterilization by filtration through a 0.45 μM microporous filter membrane, sucrose was added as a protective agent, and the mixture was freeze-dried using a freeze dryer, and the lyophilized powder was collected to obtain the anhydride-modified recombinant lactoferrin lyophilized powder.

[0044] The protein freeze-drying procedure is the same as in Example 1.

[0045] Example 4: Preparation of 4-hydroxyphthalic anhydride-modified anhydride-modified recombinant lactoferrin

[0046] At 25°C, 4-hydroxyphthalic anhydride was dissolved in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; recombinant lactoferrin was dissolved in 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30 mg / ml; the above 4-hydroxyphthalic anhydride solution was added to the protein solution, stirred and mixed, the final concentration of 4-hydroxyphthalic anhydride in the reaction system was 30 mM, and the reaction was allowed to stand for 2 hours to obtain anhydride-modified recombinant lactoferrin; after sterilization by filtration through a 0.45 μM filter membrane, it was purified by ion exchange using pH 7.2 phosphate buffer; after sterilization by filtration through a 0.45 μM microporous filter membrane, glucose was added as a protective agent, and the mixture was freeze-dried using a freeze dryer. The lyophilized powder was collected to obtain the anhydride-modified recombinant lactoferrin lyophilized powder.

[0047] The protein freeze-drying procedure is the same as in Example 1.

[0048] Example 5: Preparation of 3-hydroxyphthalic anhydride-modified anhydride-modified recombinant lactoferrin

[0049] The preparation method is basically the same as in Example 1, except that the 4-hydroxyphthalic anhydride in Example 1 is replaced with 3-hydroxyphthalic anhydride to obtain anhydride-modified recombinant lactoferrin lyophilized powder.

[0050] The protein freeze-drying procedure is the same as in Example 1.

[0051] Example 6: Preparation of a bioactive protein dressing for the prevention or treatment of human papillomavirus infection

[0052] In this embodiment, the bioactive protein dressing for the prevention and treatment of human papillomavirus infection has the following components and weight percentages: 0.001% anhydride-modified recombinant lactoferrin lyophilized powder, 1% carbomer 934%, 2% glycerol, and the balance being purified water.

[0053] The preparation method includes the following steps:

[0054] 1) Add carbomer to purified water, stir, and let stand to dissolve.

[0055] 2) Dissolve the anhydride-modified recombinant lactoferrin lyophilized powder prepared in Example 1 in an appropriate amount of purified water, then add it to the solution in step 1), add glycerol and stir for 20 minutes to mix well, then add purified water and stir for 30 minutes to obtain the dressing.

[0056] Example 7: Preparation of a bioactive protein dressing for the prevention or treatment of human papillomavirus infection

[0057] In this embodiment, the bioactive protein dressing for the prevention and treatment of human papillomavirus infection has the following components and weight percentages: 0.01% anhydride-modified recombinant lactoferrin lyophilized powder, 1% carbomer 934%, 1.5% glycerol, and the balance being purified water.

[0058] The preparation method is the same as in Example 6, except that step 2) uses the anhydride-modified recombinant lactoferrin lyophilized powder prepared in Example 2.

[0059] Example 8: Preparation of a bioactive protein dressing for the prevention or treatment of human papillomavirus infection

[0060] In this embodiment, the bioactive protein dressing for the prevention and treatment of human papillomavirus infection has the following weight percentage composition: 0.1% anhydride-modified recombinant lactoferrin lyophilized powder, 1% carbomer 934, 2% glycerol, and the balance being purified water.

[0061] The preparation method is the same as in Example 6, except that step 2) uses the anhydride-modified recombinant lactoferrin lyophilized powder prepared in Example 3.

[0062] Example 9: Determination of the concentration of anhydride-modified recombinant lactoferrin

[0063] The concentration of the anhydride-modified recombinant lactoferrin, which was not lyophilized after the first purification in Examples 1-5, was determined according to the operating procedures of the BCA protein concentration analysis kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.). The specific method is as follows:

[0064] 1) Dilute the BSA protein standard to a final concentration of 0, 0.0625, 0.125, 0.25, 0.5, 1, 2 ug / uL. The dilution of the standard is the same as that of the protein sample solution to be tested.

[0065] 2) Add 100 μL each of the diluted BSA standard and the protein sample to be tested (stock solution or dilution) to the labeled test tubes.

[0066] 3) Prepare the reaction working solution by mixing BCA-A (solution A) and BCA-B (solution B) at a volume ratio of 50:1 and mixing thoroughly.

[0067] 4) Add 2 ml of BCA working solution to each test tube, mix thoroughly, incubate in a 37°C water bath for 30 minutes, cool each tube to room temperature, and complete the test within 3-5 minutes.

[0068] 5) Measure the absorbance of each sample and BSA standard at 562 nm using a spectrophotometer and record the results. Plot a standard curve and calculate the protein concentration in the samples.

[0069] The concentrations of the proteins from Examples 1-5 were determined using the methods described above, and the results are shown in Table 1.

[0070] Table 1: Protein concentration after purification

[0071] Protein concentration mg / ml Example 1 25.03-25.54 mg / ml Example 2 25.87-26.39 mg / ml Example 3 26.08-26.73 mg / ml Example 4 24.96-25.61 mg / ml Example 5 25.42-26.11 mg / ml

[0072] The results above show that ultrafiltration or ion exchange purification can yield proteins with higher concentrations suitable for lyophilization.

[0073] Example 10: Detection of Zeta Potential of Anhydride-Treated Recombinant Lactoferrin

[0074] The lyophilized samples from Examples 1-5 were prepared into protein solutions of 1 mg / ml, and their zeta potentials were measured using a MALVERN ZEN 3600 instrument. The protein detection results from Examples 1-5 are shown in Table 2.

[0075] Table 2: Potential of anhydride-modified recombinant lactoferrin

[0076] Zeta potential (Mv) Example 1 -45.2～-51.6 Example 2 -43.1～-48.6 Example 3 -44.6～-49.2 Example 4 -42.3～-47.9 Example 5 -41.6～-45.7

[0077] The potential of recombinant lactoferrin without anhydride modification is typically 4.8 Mv.

[0078] The results above show that recombinant lactoferrin can be modified with 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride anhydride to obtain more negative charge and lower potential.

[0079] Example 11 Stability test of anhydride-modified recombinant lactoferrin

[0080] The anhydride-modified recombinant lactoferrin from Example 1 was stored for 12 months after lyophilization and without lyophilization, and the protein concentration was measured. The results are as follows:

[0081] Table 3: Protein Concentration Detection Results

[0082] Protein concentration mg / ml Acid anhydrous recombinant lactoferritin lyophilized powder after standing 23.78～24.65 Acid anhydrous recombinant lactoferritin not lyophilized after standing 5.26～6.04

[0083] The results above show that the concentration of anhydride-modified recombinant lactoferrin remained essentially unchanged after 12 months of storage using the freeze-drying method, indicating better stability.

[0084] The anhydride-modified recombinant lactoferrin from Example 1 was stored as lyophilized powder and non-lyophilized sample for 12 months, respectively, and then prepared into a 1 mg / ml protein solution. Potentiometric detection was performed, and the results are as follows:

[0085] Table 4: Protein potential detection results

[0086] Zeta potential (Mv) Acid anhydrous recombinant lactoferritin lyophilized powder after standing -44.8～-50.8 Acid anhydrous recombinant lactoferritin not lyophilized after standing -38.2～-43.2

[0087] The results above show that after storing anhydride-modified recombinant lactoferrin for 12 months using the freeze-drying method, the potential remained basically unchanged, indicating better stability.

[0088] Example 12 Comparison of modifications of bovine lactoferrin and recombinant lactoferrin

[0089] Following the method in Example 1, bovine lactoferrin and recombinant lactoferrin were modified by anhydride conversion with 4-hydroxyphthalic anhydride. The resulting lyophilized powders were then prepared into 1 mg / ml protein solutions, and potential detection was performed. The results are as follows: Figure 1 and Figure 2 As shown.

[0090] from Figure 1 As can be seen, bovine lactoferrin modified with 4-hydroxyphthalic anhydride exhibits more impurity peaks, and the zeta potential of the lyophilized powder is -34 Mv.

[0091] from Figure 2 As can be seen, the recombinant lactoferrin modified by anhydride with 4-hydroxyphthalic anhydride has fewer impurity peaks, almost none, and the resulting lyophilized powder has a negative charge of -51.6 Mv.

[0092] Conclusion: The recombinant lactoferrin modified with 4-hydroxyphthalic anhydride has fewer impurity peaks, more negative charge, and lower potential.

[0093] Example 13: Determination of the antibacterial activity of recombinant lactoferrin and anhydride-modified recombinant lactoferrin

[0094] The bacterial strain was inoculated into broth peptone medium and cultured for 16–20 h. Bacterial counts were performed on nutrient agar plates, and the bacterial suspension concentration was adjusted to 10⁶–10⁸ CFU / ml. The juvenile bacterial suspension of the above strain was inoculated onto nutrient agar medium and spread evenly. A sterile Oxford cup was placed in the center of the petri dish, and 0.1 ml of sterile microfiltered lactoferrin at different dilutions and the anhydride-modified lactoferrin solution from Example 1 (pH 7.0) were added to the cup, with sterile water as a control. After incubation at 37°C for 24 h, the diameter of the inhibition zone (mm) was measured. The lowest sample concentration with an inhibition zone diameter larger than the control group (8 mm) was defined as the minimum inhibitory concentration (MIC, μg / ml).

[0095] Table 5. Diameter of the inhibition zone (mm) produced by recombinant lactoferrin against different microorganisms.

[0096]

[0097] Table 6. Diameter of inhibition zones (mm) produced by the antibacterial effects of anhydride-modified recombinant lactoferrin on different microorganisms.

[0098]

[0099] It can be found that recombinant lactoferrin itself has broad-spectrum antibacterial and antioxidant effects. It has varying degrees of inhibitory and killing effects on many microorganisms, including Gram-positive and Gram-negative aerobic and anaerobic bacteria and some fungi. Even after being modified by acid anhydride modification with 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride, it still maintains good antibacterial effects.

[0100] Example 14: Detection of the anti-HPV activity of anhydride-modified recombinant lactoferrin

[0101] (1) Culture HeLa cells: The cultured HeLa cells (purchased from Beina Chuanglian Biotechnology Co., Ltd.) were evenly spread in a 60mm cell culture dish;

[0102] (2) The anhydride-modified recombinant lactoferrin lyophilized powder obtained in Example 1 was mixed with pure water to prepare concentrations of 1 mg / ml, 0.5 mg / ml, and 0.25 mg / ml;

[0103] (3) Sample addition: Add 3 ml of the different concentrations of anhydride-modified recombinant lactoferrin lyophilized powder prepared in step 2) to each cell culture dish;

[0104] (4) Cell collection: Cells were collected every 24 hours, and total cell protein was extracted. Cells were also collected every 48 hours, and total cell protein was extracted.

[0105] (5) Protein immunoblotting analysis: Shanghai, gel running, membrane transfer, and incubation with Papillomavirus 16E7 antibody (purchased from Abcam) and Anti HPV 18E7 antibody (purchased from Abcam), respectively.

[0106] The results are as follows Figure 3 and Figure 4 As shown.

[0107] Figure 3 These are the results of the anti-HPV 16E7 antibody test. Among them, 1 represents normal HeLa cells; 2 represents total cell protein from a 1 mg / ml sample over 24 hours; 3 represents total cell protein from a 1 mg / ml sample over 48 hours; 4 represents total cell protein from a 0.5 mg / ml sample over 24 hours; 5 represents total cell protein from a 0.5 mg / ml sample over 48 hours; 6 represents total cell protein from a 0.25 mg / ml sample over 24 hours; and 7 represents total cell protein from a 0.25 mg / ml sample over 48 hours.

[0108] Figure 4The results are for the detection of Anti-HPV 18E7 antibody. Among them, 1 represents normal HeLa cells; 2 represents total cell protein in cells after 24 hours of a 1 mg / ml sample; 3 represents total cell protein in cells after 48 hours of a 1 mg / ml sample; 4 represents total cell protein in cells after 24 hours of a 0.5 mg / ml sample; 5 represents total cell protein in cells after 48 hours of a 0.5 mg / ml sample; 6 represents total cell protein in cells after 24 hours of a 0.25 mg / ml sample; and 7 represents total cell protein in cells after 48 hours of a 0.25 mg / ml sample.

[0109] It can be seen that after reacting with 1 mg / ml of anhydride-modified recombinant lactoferrin lyophilized powder for 24 hours, the expression of HPV 16E6 protein and HPV 18E7 protein could not be detected. This indicates that after adding 1 mg / ml of anhydride-modified recombinant lactoferrin lyophilized powder, HPV viral capsid protein could not be synthesized normally, suggesting that anhydride-modified recombinant lactoferrin has an inhibitory effect on HPV 16 and HPV 18.

[0110] Example 15 Comparison of lysine and arginine residue modification rates in 4-hydroxyphthalic anhydride-modified recombinant lactoferrin, 3-hydroxyphthalic anhydride-modified recombinant lactoferrin, and 4-hydroxyphthalic anhydride-modified lactoferrin.

[0111] The ratio of lysine residues at the ends of the modified protein to the corresponding acid anhydride was detected using TNBS; the ratio of arginine residues at the ends of the modified protein to the corresponding acid anhydride was detected using p-HPG.

[0112] Table 7. Results of lysine and arginine modification rates in 4-hydroxyphthalic anhydride-modified recombinant lactoferrin, 3-hydroxyphthalic anhydride-modified recombinant lactoferrin, and 4-hydroxyphthalic anhydride-modified lactoferrin.

[0113]

[0114] It can be seen that the lysine and arginine modification effects of recombinant lactoferrin modified with 4-hydroxyphthalic anhydride and 3-hydroxyphthalic anhydride are better than those of lactoferrin modified with 4-hydroxyphthalic anhydride.

[0115] Example 16 Comparison of antiviral activities of 4-hydroxyphthalic anhydride-modified recombinant lactoferrin, 3-hydroxyphthalic anhydride-modified recombinant lactoferrin, and 4-hydroxyphthalic anhydride-modified lactoferrin.

[0116] 1. Constructing HPV fake viruses

[0117] The HPV L2-related gene was codon-optimized, amplified using polymerase chain reaction (PCR), cloned into a eukaryotic expression vector, and then identified by restriction enzyme digestion and sequencing to form stable expression vectors for pHPV6, pHPV16, and pHPV18. The three expression vectors and reporter plasmid pN31-EGFP, which were correctly identified by sequencing, were amplified in large quantities for later use. All expression vectors and reporter plasmids used were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0118] 293FT cells (purchased from Beina Chuanglian Biotechnology Co., Ltd.) were seeded in 96-well cell culture plates. After 12 hours, pHPV6, pHPV16, pHPV18 expression plasmids and pN31-EGFP expression plasmid were mixed in proportion and added to 0.25 mol / L CaCl2 solution in different amounts. The CaCl2 solution of the plasmids was added dropwise to an equal volume of 2*HeBS (pH=6.97) solution, mixed thoroughly, and allowed to stand for 5 minutes. The mixture was then added to the pre-seeded 293FT cell wells. After 12 hours, the culture medium was changed. 48 hours after transfection, cells from each well were collected and lysed with the same volume of lysis buffer at 37°C for 16 hours. The supernatant was collected as the pseudovirus suspension, aliquoted, and stored at -80°C for later use.

[0119] 2. Virus titer determination

[0120] The HPV pseudovirus suspension was repeatedly diluted 10-fold, from 10... -1 Up to 10 -6 Remove the culture medium from the 96 wells, add 50 μL of pseudovirus diluent, then add 5 μL of normal culture medium, incubate at 37°C with 5% CO2. 2 Continue culturing for 72 hours, take 50 μL of culture supernatant, and use the Merck alkaline phosphatase assay kit to detect it, and calculate TCID50 accordingly.

[0121] Inhibitory activity of recombinant lactoferrin modified with 4-hydroxyphthalic anhydride (prepared in Example 1), recombinant lactoferrin modified with 3-hydroxyphthalic anhydride (prepared in Example 5), and lactoferrin modified with 4-hydroxyphthalic anhydride (prepared in Example 12) against HPV6, HPV16, and HPV18 was detected.

[0122] 293FT cells were passaged into 96-well plates with 1 x 10⁵ cells per well and incubated at 5% CO₂. 2The culture was carried out for 8 hours. 100 TCID50 of HPV6, HPV16, and HPV18 pseudoviruses (50 M) were incubated with different concentrations of lyophilized 4-hydroxyphthalic anhydride-modified recombinant lactoferrin powder solutions prepared according to Example 1, as well as lyophilized 3-hydroxyphthalic anhydride-modified recombinant lactoferrin powder solutions, and lyophilized 4-hydroxyphthalic anhydride-modified lactoferrin powder solutions, for a total of three solutions, at 37°C for 30 min. The culture medium in the 96-well plate was removed, and the incubated mixture was added. The plate was then incubated at 37°C with 5% CO2 for another 12 hours. Fresh culture medium was then added, and after 72 hours of incubation, the supernatant of 50 M culture medium was collected and analyzed using a Merck alkaline phosphatase assay kit. The IC50 (half-maximal inhibitory concentration) of 4-hydroxyphthalic anhydride-modified recombinant lactoferrin, 3-hydroxyphthalic anhydride-modified recombinant lactoferrin, and 4-hydroxyphthalic anhydride-modified lactoferrin was calculated using SPSS 25. The results are shown in Table 8.

[0123] Table 8. Anti-HPV activity of 4-hydroxyphthalic anhydride-modified recombinant lactoferrin, 3-hydroxyphthalic anhydride-modified recombinant lactoferrin, and 4-hydroxyphthalic anhydride-modified lactoferrin.

[0124]

[0125] It can be seen that the recombinant lactoferrin modified with 4-hydroxyphthalic anhydride and 3-hydroxyphthalic anhydride has a better inhibitory effect on HPV6, HPV16 and HPV18 than the lactoferrin modified with 4-hydroxyphthalic anhydride.

[0126] Example 17

[0127] The efficacy of the dressing for the prevention and treatment of human papillomavirus infection of the present invention was tested. Details are as follows:

[0128] Methods and Materials

[0129] Patients who visited Taizhou People's Hospital between July 2021 and June 2022 were included in this study. 240 patients with high-risk HPV infection and cervical cytological abnormalities were selected and randomly divided into an experimental group (n=120, aged 18-59 years) and a control group (n=120, aged 18-60 years). There were no statistically significant differences in age or the rate of cervical cytological abnormalities between the two groups.

[0130] Inclusion criteria

[0131] (1) Women aged 18 or older who voluntarily participate in this study

[0132] (2) Women who have had sexual intercourse

[0133] (3) Patients diagnosed with cervicitis

[0134] (4) Patients diagnosed with cervical intraepithelial neoplasia by microscopy and biopsy

[0135] (5) Patients who test positive for high-risk HPV-DNA

[0136] (6) Patients who have not received any treatment in the past three months

[0137] Exclusion criteria:

[0138] (1) Pregnant and lactating women

[0139] (2) Acute reproductive tract inflammation

[0140] (3) Malignant tumors and other diseases that affect the body's immune status.

[0141] The experimental group received vaginal administration of the anti-HPV anhydride lactoferrin gel (3g / vial) prepared in Example 8, one vial each time, once every other day (used before bedtime), for 9 times as one course of treatment, for a total of 3 courses. Avoid use during menstruation.

[0142] The control group received an anti-HPV vaginal liquid dressing (3g / vial) prepared from anhydride-modified lactoferrin according to patent CN112999358A, administered vaginally once every other day (before bedtime), for 9 times as one course of treatment, for a total of 3 courses. Avoid use during menstruation.

[0143] Both groups underwent cervical cytology, HPV testing, and examination for abnormal vaginal discharge and odor at the same time.

[0144] Experimental results:

[0145] 2.1 HPV infection typing of the two groups of patients, see Table 9 for details.

[0146] Table 9. HPV infection status (n) in the two groups of patients.

[0147] Group Type 16 Type 18 Type 58 Type 56 Other subtypes Test group 36(19) 17(9) 21(11) 19(9) 27(6) Control group 30(13) 18(10) 22(4) 18(5) 32(12)

[0148] Note: The numbers in parentheses in the table represent the number of cases co-infected with other types of viruses.

[0149] 2.2 Comparison of HPV clearance rates between the two groups of patients

[0150] Three months after treatment, a total of 240 patients from both groups were followed up, and no cases were lost to follow-up. The HPV clearance rate in the study group (95.00%) was significantly different from that in the control group (55.83%) (P<0.05), as detailed in Table 10.

[0151] Table 10 Comparison of HPV clearance rates between the two groups of patients (n, %)

[0152] Group Number of cases Cure Effective Ineffective Total effective rate (%) Test group 120 71 43 6 95.00 Control group 120 42 25 53 55.83

[0153] 2.3 Comparison of cervical cytology negative conversion rates between the two groups of patients

[0154] In the study group, 47 patients had HPV combined with ASCUS / LSIL cervical cytology, while in the control group, 43 patients had HPV combined with ASCUS / LSIL cervical cytology. Six months after treatment, the negative conversion rate of cervical cytology in the experimental group (85.11%) was significantly higher than that in the control group (53.49%), with a statistically significant difference (P < 0.05). See Table 11 for details.

[0155] Table 11 Comparison of negative TCT test results between the two groups (n, %)

[0156] Group Number of cases Negative conversion cases Negative conversion rate (%) Test group 47 40 85.11 Control group 43 23 53.49

[0157] Table 12 shows two groups with abnormally reduced vaginal discharge.

[0158] Group Number of cases Significant improvement Improvement No change Total effective rate (%) Test group 120 56 50 14 88.33 Control group 120 37 26 57 52.50

[0159] In the experimental group, a total of 106 cases showed improvement in abnormal vaginal discharge, while in the control group, a total of 63 cases showed improvement. The difference between the two groups was statistically significant (P < 0.05).

[0160] Changes in vaginal discharge odor before and after treatment are detailed in Table 13:

[0161] Table 13 Changes in vaginal discharge odor before and after treatment

[0162] Group Number of cases Recovery Marked effect General Ineffective Total effective rate (%) Test group 120 61 35 14 10 91.67 Control group 120 41 21 9 49 59.17

[0163] In the experimental group, a total of 110 cases showed improvement in vaginal discharge odor, with a total effective rate of 91.67%. In the control group, a total of 71 cases showed improvement in vaginal discharge odor, with a total effective rate of 59.17%. The difference between the two groups was statistically significant (P < 0.05).

[0164] 2.4 Adverse Reactions

[0165] No adverse reactions occurred in the experimental group during medication, and patient compliance was good. No cases of pathological grade worsening were found in patients requiring repeat testing.

[0166] Clinical trial conclusions: The anti-HPV anhydride-modified lactoferrin gel involved in this invention is significantly effective in treating high-risk HPV infection, significantly improving clinical symptoms, especially for excessive discharge, foul-smelling discharge, postcoital bleeding, and cervical erosion. Most importantly, it significantly increases the HPV seroconversion rate. Clinical application is safe and reliable, with no significant toxic side effects or adverse reactions. The gel provides a protective barrier, preventing bacterial infection and other external irritants, and promoting wound healing. The anti-HPV anhydride-modified lactoferrin gel can improve the vaginal microenvironment, reduce vaginal inflammation, and efficiently and rapidly kill HPV non-specifically without causing viral mutations or changes in the vaginal environment. It eliminates HPV infection, prevents cervical intraepithelial neoplasia and cervical cancer, and improves local immune function.

[0167] Example 17 Safety Testing

[0168] The dressing of Example 6 was tested for safety according to the method specified in GB / T1688.10-2017. The dressing showed a cytotoxic cell survival rate of 92.2%, a vaginal irritation index of 2.8, and no skin sensitization reaction.

[0169] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A recombinant bioactive protein for the prevention or treatment of human papillomavirus infection, characterized in that, The recombinant bioactive protein is anhydride-modified recombinant lactoferrin, anhydride-modified recombinant lactoglobulin, or anhydride-modified recombinant histone. The recombinant bioactive protein is obtained by anhydride modification of recombinant lactoferrin, recombinant lactoglobulin, or recombinant histone with 3-hydroxyphthalic anhydride or its derivatives, 4-hydroxyphthalic anhydride or its derivatives, succinic anhydride, or maleic anhydride.

2. The recombinant bioactive protein according to claim 1, characterized in that, The recombinant bioactive protein is an anhydride-modified recombinant lactoferrin modified with 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride.

3. The recombinant bioactive protein according to claim 1, characterized in that, The recombinant bioactive protein is an anhydride-modified recombinant lactoglobulin modified with 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride.

4. The method for preparing the recombinant bioactive protein according to claim 2, wherein the recombinant bioactive protein is anhydride-modified recombinant lactoferrin modified with 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride, characterized in that... The process includes the following steps: 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride is dissolved in dimethyl sulfoxide at a temperature of 25-50℃ to obtain a saturated 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride solution; recombinant lactoferrin is dissolved in 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30-80 mg / ml; the above 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride solution is added to the protein solution and stirred until homogeneous, with a final concentration of 30-80 mM in the reaction system; the mixture is allowed to stand for 2 hours to obtain anhydride-modified recombinant lactoferrin; after sterilization by filtration through a 0.45 μm filter membrane, ultrafiltration or ion exchange purification is performed using pH 7.2 phosphate buffer; after sterilization by filtration through a 0.45 μm microporous filter membrane, a protective agent is added, and the mixture is freeze-dried using a freeze dryer. The lyophilized powder is collected to obtain the anhydride-modified recombinant lactoferrin lyophilized powder.

5. The method for preparing recombinant bioactive protein according to claim 4, characterized in that, The protein freeze-drying process includes the following stages: pre-freezing stage: the temperature of the separator is lowered to -80°C and held for 6 hours; first sublimation stage: the temperature of the separator is raised to -25°C and held for 12 hours; second drying stage: the temperature of the separator is raised to 20°C and held for 8 hours. The freeze-drying protectant is mannitol, sucrose, glucose, trehalose, or fructose.

6. The use of the recombinant bioactive protein as described in any one of claims 1-3 in the preparation of drugs for the prevention or treatment of human papillomavirus infection.

7. A pharmaceutical formulation containing the recombinant bioactive protein as described in any one of claims 1-3, characterized in that, The formulation contains a pharmaceutically effective dose of recombinant bioactive protein and pharmaceutically acceptable excipients or auxiliary ingredients.

8. The pharmaceutical preparation according to claim 7, characterized in that, The preparation is a liquid preparation, ointment, gel, or suppository.

9. The pharmaceutical preparation according to claim 8, characterized in that, The formulation is a dressing, and the recombinant bioactive protein in the formulation is the 3-hydroxyphthalic anhydride or 4-hydroxyphthalic anhydride-modified recombinant lactoferrin as described in claim 2. The components and their weight percentages of the dressing are as follows: 0.001-0.1% lyophilized recombinant lactoferrin powder, 1-2% carbomer 934, 0.5%-2% glycerol, and the balance being purified water.

10. The pharmaceutical preparation according to claim 9, characterized in that, The method for preparing the dressing includes the following steps: 1) Dissolve carbomer in purified water by stirring and letting it stand. 2) Dissolve the anhydride-modified recombinant lactoferrin in an appropriate amount of purified water, then add it to the solution in step 1), add glycerol and stir for 20-60 minutes to mix well, then add purified water and stir for 30 minutes to obtain the dressing.

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