Anti-HPV infection recombinant humanized collagen composition and preparation method thereof

Abstract

The invention discloses an anti-HPV (human papillomavirus) infection recombinant humanized collagen composition and a preparation method thereof, and the composition comprises the following components in percentage by mass: 30%-45% of recombinant humanized collagen fusion protein, 30%-40% of probiotics and 20%-35% of glycogen, the recombinant humanized collagen fusion protein comprises a recombinant humanized collagen fragment and a humanized fibronectin fragment which are sequentially connected from an N end to a C end, the recombinant humanized collagen fragment and the humanized fibronectin fragment are connected by using a linker. The recombinant humanized collagen fusion protein in the composition can promote the adhesion and colonization of the probiotic composition in vaginal epithelial cells, rapidly improve the microbial environment of the vagina, and effectively enhance the resistance of the vagina to harmful bacteria and viruses.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a recombinant humanized collagen composition for resisting HPV infection and a preparation method thereof. Background Art

[0002] HPV is a DNA virus that can exist in two forms: free or integrated. It replicates and transcribes in host cells to achieve persistent viral infection and cause cell tumorigenesis. The persistence of HPV infection is affected by the surrounding microenvironment. The local microenvironment of the cervix plays an important role in maintaining tissue homeostasis, resisting pathogens, and participating in immune responses, and can affect the natural history of HPV infection. The vaginal microbiota (VMB) is an important part of the cervical microenvironment. Its specific characteristics are closely related to gynecological diseases and reproductive health. The composition of VMB can affect the local immune response. VMB dominated by a certain type of lactobacillus can reduce the risk of HPV infection, participate in the clearance of HPV infection, and prevent the occurrence of cervical cancer. In recent years, the role of changes in vaginal microecology in genital HPV infection has attracted the attention of more and more scholars. Researchers have used high-throughput sequencing of vaginal secretions of normal women with negative HPV and vaginal secretions of HPV-positive women and found that there are obvious differences in the vaginal microecology between the two. HPV-positive women mainly show highly diverse vaginal microorganisms, with a decrease in lactobacilli and an increase in vaginal Gardnerella. In addition, a large number of literatures have shown that bacterial vaginosis (BV) is positively correlated with the incidence, prevalence, and persistence of HPV infection. BV is actually a vaginal flora disorder, the main phenotype of which is the reduction of lactobacilli that secrete H2O2 in the vagina, and the proliferation of Gardnerella and anaerobic bacteria. Under normal circumstances, lactobacilli prevent the colonization of bacterial species associated with bacterial vaginosis by maintaining low pH and producing bacteriocins, which is important for maintaining the cervical epithelial barrier function that inhibits HPV from entering basal keratinocytes. When strict anaerobes associated with BV are able to colonize, they produce enzymes and metabolites, which may damage this barrier and provide a good environment for HPV infection and invasion. The entry of HPV, especially the integration of high-risk HPV fragments and host genes and ultimately tumor transformation. Therefore, HPV infection is inseparable from the disorder of the local microecological environment of the vagina, especially the reduction of lactobacilli.

[0003] A large number of studies have confirmed that vaginal administration of probiotics can effectively treat and prevent HPV infection, and some products have been approved for marketing. However, the current course of vaginal administration of probiotics is relatively long. The reason is that probiotics do not quickly and effectively colonize on the surface of vaginal mucosal epithelial cells, resulting in the failure to fully exert their efficacy and a large amount of active ingredients are excreted from the body, causing waste.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention provides a recombinant humanized collagen composition for anti-HPV infection and its preparation method. The composition includes a recombinant humanized collagen fusion protein, glycogen, and a probiotic composition. The recombinant humanized collagen fusion protein can enhance the adhesion of beneficial bacteria to epithelial cells, promote their reproduction, maintain the stability of the vaginal microbiota, enhance the resistance of the vagina to harmful bacteria and viruses, and reduce the risk of HPV infection.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, the present invention provides a recombinant humanized collagen composition for anti-HPV infection, which includes the following components by mass percentage: 30% - 45% of recombinant humanized collagen fusion protein, 30% - 40% of probiotics, and 20 - 35% of glycogen; the recombinant humanized collagen fusion protein includes a recombinant humanized collagen fragment and a human fibronectin fragment connected in sequence from the N-terminus to the C-terminus; a linker is used to connect the recombinant humanized collagen fragment and the human fibronectin fragment; the amino acid sequence of the recombinant humanized collagen fragment is as shown in SEQ ID NO.2; the amino acid sequence of the human fibronectin fragment is as shown in SEQ ID NO.3.

[0008] In some embodiments, the linker is GGGGGG.

[0009] In some embodiments, the amino acid sequence of the recombinant humanized collagen fusion protein is as shown in SEQ ID NO.1.

[0010] In some embodiments, the recombinant humanized collagen fusion protein further includes a HIS tag fragment, preferably HHHHHH.

[0011] SEQ ID NO.1:

[0012] GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGGGGGSVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT

[0013] SEQ ID NO.2:

[0014] GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR

[0015] SEQ ID NO.3:

[0016] VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKST ATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT

[0017] In the present invention, SEQ ID NO.2 is a triple repeat sequence of SEQ ID NO.4, and SEQ ID NO.4 is screened and optimized from the human native type III collagen COL-III sequence. The sequence of the human collagen type III is the NCBI reference sequence: KAI4037253.1, see https: / / www.ncbi.nlm.nih.gov / protein / KAI4037253.1. Through a large number of studies, the applicant found that the selected sequence has strong water solubility, high recombinant expression yield, simple purification process, and better biological activity than other sequences of commercialized collagen.

[0018] SEQ ID NO.4:

[0019] GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR

[0020] SEQ ID NO.3 is a fragment of human fibronectin (FN). Human fibronectin is a glycoprotein in the extracellular matrix. Fibronectin can bind to extracellular matrix components such as collagen, fibrin, and heparan sulfate proteoglycans (such as syndecan) and participate in cell adhesion, cell movement, opsonization, wound healing, and maintenance of cell shape. In addition, fibronectin can bind to the integrin αvβ3 on the cell surface to enhance cell adhesion. The selected SEQ ID NO.3 fragment in the present invention has a good fusion effect with SEQ ID NO.2, that is, it has high expression activity in competent cells, and the obtained fusion protein can effectively improve the adhesion of probiotics to vaginal epithelial cells.

[0021] In some embodiments, the nucleotide sequence of the recombinant humanized collagen fusion protein is as shown in SEQ ID NO.5.

[0022] SEQ ID NO.5

[0023] GGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGTGGTGGTGGTGGTTCTGTCTCAGATGTACCCAGGGACCTAGAAGTTGTCGCAGCGACCCCGACCAGCTTGCTGATTTCCTGGGATGCGCCTGCGGTTACCGTTCGTTATTACCGTATTACCTATGGTGAGACAGGCGGAAACAGCCCGGTTCAAGAATTCACCGTGCCAGGTTCGAAAAGCACCGCAACGATTTCTGGCCTGAAACCGGGCGTCGATTATACCATCACCGTTTACGCAGTTACGGGTAGAGGCGACAGCCCAGCGTCTTCCAAACCGATTAGTATTAACTACCGCACC

[0024] In some embodiments, the probiotics include at least two of Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus rhamnosus, and Lactobacillus reuteri.

[0025] In some embodiments, the probiotics are a mixed bacterium of Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus rhamnosus, and Lactobacillus reuteri mixed at a viable cell count ratio of 1:0.2:0.4:0.2.

[0026] In some embodiments, the total viable cell count in the recombinant humanized collagen composition against HPV infection is 10 6 ~10 9 CFU / g.

[0027] In some embodiments, the glycogen includes at least one of β-glucan, isomaltooligosaccharide, inulin, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, stachyose, mannan oligosaccharide, and arabinan oligosaccharide.

[0028] In some embodiments, the method for preparing the recombinant humanized collagen fusion protein includes the following steps:

[0029] Construct an expression vector containing a nucleic acid molecule encoding the recombinant humanized collagen fusion protein in any of the above embodiments, transfer the expression vector into a recipient cell, culture the recipient cell, induce expression, break the bacterial cells, and perform purification to obtain the recombinant humanized collagen fusion protein.

[0030] In some embodiments, the method for preparing the recombinant humanized collagen fusion protein further includes mixing the recombinant humanized collagen fusion protein with a stabilizer and freeze-drying, specifically including: adding a stabilizer to a PBS solution (pH = 6.4 - 7.4) of the recombinant humanized collagen fusion protein with a mass percentage of 0.01% - 0.5% to obtain a freeze-drying solution, and then performing freeze-drying to obtain the freeze-dried powder of the recombinant humanized collagen fusion protein.

[0031] In some embodiments, the stabilizer includes glycine, trehalose, mannitol, and Tween 80. The mass percentage of glycine in the freeze-drying solution is 1% - 2%, the mass percentage of trehalose in the freeze-drying solution is 0.5% - 1.5%, the mass percentage of mannitol in the freeze-drying solution is 2% - 4%, and the mass percentage of Tween 80 in the freeze-drying solution is 0.001% - 0.004%.

[0032] In some embodiments, the freeze-drying conditions include: pre-freezing at -20°C for 6 - 9 h, and then performing vacuum freeze-drying at -40°C for 24 - 48 h.

[0033] Second aspect, the present invention provides a method for preparing the recombinant humanized collagen composition for anti-HPV infection in any of the above embodiments, which is characterized by comprising the following steps:

[0034] (1) Obtain solid recombinant humanized collagen fusion protein, solid probiotic and glycogen; preferably, the solid recombinant humanized collagen fusion protein is the freeze-dried powder of the recombinant humanized collagen fusion protein; the solid probiotic is the freeze-dried powder of the probiotic;

[0035] (2) Mix the solid recombinant humanized collagen fusion protein, the solid probiotic and the glycogen.

[0036] Third aspect, the present invention provides a preparation comprising the composition in any of the above embodiments, including but not limited to capsules, gels, tablets, suppositories, creams or effervescent tablets.

[0037] Fourth aspect, the present invention provides a vaginal gel, which comprises the following components by mass percentage: 2% - 6% of the recombinant humanized collagen composition, 8% - 15% of the gel matrix, 1% - 3% of the thickening agent, 3% - 10% of the humectant, 1% - 2% of the plant extract, 0 - 1% of the pH regulator, and the balance being water and PBS buffer.

[0038] In some embodiments, the gel matrix is selected from at least one of carbomer, xanthan gum, poloxamer and gelatin.

[0039] In some embodiments, the thickening agent is selected from at least one of carboxymethyl cellulose and its salts, hydroxyethyl cellulose, and hydroxypropyl cellulose.

[0040] In some embodiments, the humectant is selected from at least one of glycerol, propylene glycol, butylene glycol, hyaluronic acid and its salts.

[0041] In some embodiments, the plant extract is at least one of tea tree essential oil, green tea extract, chamomile extract, and aloe extract.

[0042] In some embodiments, the pH regulator is at least one of lactic acid and citric acid.

[0043] The present invention has the following beneficial effects:

[0044] The present invention provides a recombinant humanized collagen composition for anti-HPV infection. The recombinant humanized collagen fusion protein in the composition can promote the adhesion and colonization of the probiotic composition on vaginal epithelial cells, rapidly improve the vaginal microbial environment, and effectively enhance the resistance of the vagina to harmful bacteria and viruses.

[0045] The present invention provides a recombinant humanized collagen composition for anti-HPV infection. When this composition is applied to a vaginal gel, the probiotics therein can still maintain relatively high activity, and it has preventive and therapeutic effects on HPV infection, as well as good moisturizing properties. Brief Description of the Drawings

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0047] Figure 1 Electrophoresis diagrams of the recombinant humanized collagen fusion protein prepared in Example 1 and the recombinant humanized collagen fragment prepared in Comparative Example 1. Detailed Embodiments

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0049] In the description of the embodiments of the present application, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity, specific order, or primary and secondary relationship of the indicated technical features.

[0050] When "embodiment" is mentioned herein, it means that the specific features, structures, or characteristics described in connection with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various positions in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments.

[0051] In the embodiments of the present application, the term "or / and" is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A or / and B can represent: A exists alone, A and B exist simultaneously, and B exists alone.

[0052] In addition, the character " / " in this article generally represents an "or" relationship between the associated objects before and after.

[0053] In the embodiments of the present application, the meaning of "a plurality of" is two or more (including two). Similarly, "a plurality of groups" means two or more groups (including two groups), and "a plurality of layers" means two or more layers (including two layers), unless otherwise clearly specified and limited.

[0054] In the embodiments of the present application, "at least one" means one or more than one.

[0055] In the embodiments of the present application, the orientations or positional relationships indicated by the technical terms "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the embodiments of the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed in a specific orientation, etc., and cannot be understood as limiting the embodiments of the present application. Those skilled in the art can understand the specific meanings of the above terms in the embodiments of the present application according to specific circumstances.

[0056] Table 1 is a description of the sources of raw materials used in some specific implementation methods.

[0057] Table 1

[0058]

[0059] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0060] Example 1

[0061] A recombinant humanized collagen composition for resisting HPV infection comprises the following components calculated by mass percentage: 40% recombinant humanized collagen fusion protein, 35% probiotic composition, and 25% glycogen.

[0062] Among them, the preparation method of recombinant humanized collagen fusion protein includes:

[0063] (1) The nucleotide sequence of SEQ ID NO.5 was cloned into the prokaryotic expression vector pET22b (the plasmid was synthesized and constructed by Suzhou Hongxun Biotechnology Co., Ltd.); the plasmid vector pET22b has a 6*His tag. The synthesized plasmid was then transformed into E. coli BL21 competent cells.

[0064] (2) Positive recombinants were screened on ampicillin-resistant plates, and then the strains with correct sequencing were transferred to TB medium containing ampicillin resistance (50 ng / μL) and cultured overnight at 37°C with shaking. The next day, the activated strains were inoculated into 1 L of fresh TB medium for large-scale culture at a ratio of 1:100. When the OD600 reached 0.6 at 37°C with shaking at 180 rpm, the temperature was lowered to 18°C, and isopropyl β-D-thiogalactopyranoside (IPTG) was added. The final concentration of IPTG was 1 mM, and induction expression was continued for 18 h under the same conditions, and then the bacterial solution was collected. The bacteria were collected by centrifugation at 5000 rpm for 30 min at 4°C, resuspended and washed with 50 mL of ultrapure water ddH2O, and then centrifuged again to collect the bacteria. The collected bacteria were resuspended in 50 mL of ddH2O, and then the bacteria were lysed using a high-pressure cell disruptor at a pressure of 800 Pa for 10 min at 4°C. The lysed solution was centrifuged at 10000 rpm for 1 h at 4°C, the precipitate was discarded, and the supernatant was collected for subsequent purification.

[0065] (3) After the supernatant was adsorbed by a nickel ion column, it was washed with 20 mM PBS + 200 mM NaCl + 50 mM imidazole, an aqueous solution with pH = 7.4 and 20 mM PBS + 200 mM NaCl + 100 mM imidazole, an aqueous solution with pH = 7.4 in sequence to remove impurities, and then eluted with 20 mM PBS + 200 mM NaCl + 200 mM imidazole, an aqueous solution with pH = 7.4 and 20 mM PBS + 200 mM NaCl + 500 mM imidazole, an aqueous solution with pH = 7.4. The eluate was further desalted using a G25 gel column; then centrifuged at a centrifugal force of 10000 g for 10 min, the supernatant was collected and concentrated using a 10 KD ultrafiltration tube (Millipore), centrifuged at 4500 g, ultrafiltered to 500 μL at 4°C, and the concentrate was washed three times with 1×PBS and centrifuged three times repeatedly to obtain a PBS solution (pH = 6.4) of the fusion protein with a mass percentage of 0.5%, and stored at -80°C for standby.

[0066] The gel electrophoresis results of the recombinant humanized collagen fusion protein are as Figure 1 shown. An obvious specific band can be observed at around 31 kDa, which is basically consistent with the predicted molecular weight, indicating that the target protein was successfully expressed and secreted extracellularly, and the purity reached more than 90%. Among them, lane M: protein Marker.

[0067] (4) Dissolve the stabilizer in the above-mentioned PBS solution of the fusion protein to obtain a lyophilization solution, and then perform lyophilization to obtain the freeze-dried powder of the recombinant humanized collagen fusion protein; the stabilizer includes glycine, trehalose, mannitol and Tween 80. The mass percentage of glycine in the lyophilization solution is 1.5%, the mass percentage of trehalose in the lyophilization solution is 1%, the mass percentage of mannitol in the lyophilization solution is 3%, and the mass percentage of Tween 80 in the lyophilization solution is 0.003%; the lyophilization conditions include: pre-freezing at -20°C for 8 h, and then performing vacuum freeze-drying at -40°C for 36 h.

[0068] Among them, the probiotic composition is a mixture of Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus rhamnosus and Lactobacillus reuteri mixed according to the viable bacteria number ratio of 1:0.2:0.4:0.2.

[0069] Among them, glycogen is a mixture of β-glucan, inulin and isomaltooligosaccharide mixed according to the mass ratio of 1:1:0.3.

[0070] A preparation method of a recombinant humanized collagen composition for anti-HPV infection, comprising:

[0071] Mix the freeze-dried powder of the recombinant humanized collagen fusion protein, the freeze-dried powder of the probiotic and glycogen according to the formula ratio.

[0072] Example 2

[0073] The difference from Example 1 is that the recombinant humanized collagen composition for anti-HPV infection, calculated by mass percentage, includes the following components: 35% of the recombinant humanized collagen fusion protein, 35% of the probiotic composition, and 35% of glycogen.

[0074] Example 3

[0075] The difference from Example 1 is that the recombinant humanized collagen composition for anti-HPV infection, calculated by mass percentage, includes the following components: 45% of the recombinant humanized collagen fusion protein, 35% of the probiotic composition, and 20% of glycogen.

[0076] Example 4

[0077] A recombinant humanized collagen composition for anti-HPV infection, calculated by mass percentage, includes the following components: 35% of the recombinant humanized collagen fusion protein, 30% of the probiotic composition, and 35% of glycogen.

[0078] Among them, the recombinant humanized collagen fusion protein is the same as that in Example 1;

[0079] Among them, the probiotic composition is a mixture of Lactobacillus crispatus, Lactobacillus rhamnosus and Lactobacillus reuteri mixed according to the viable bacteria number ratio of 1:0.4:0.2.

[0080] The glycogen is a mixture of β-glucan and isomaltooligosaccharide in a mass ratio of 1:1.

[0081] Example 5

[0082] A recombinant humanized collagen composition for resisting HPV infection comprises the following components calculated by mass percentage: 30% recombinant humanized collagen fusion protein, 40% probiotic composition and 30% glycogen.

[0083] Wherein, the recombinant humanized collagen fusion protein is the same as that in Example 1;

[0084] The probiotic composition is a mixture of Lactobacillus crispatus and Pediococcus acidilactici in a live bacteria count ratio of 1:0.2.

[0085] Among them, glycogen is oligofructose.

[0086] Comparative Example 1

[0087] The difference from Example 1 is that the recombinant humanized collagen fusion protein is replaced by the recombinant humanized collagen.

[0088] A recombinant humanized collagen composition for resisting HPV infection comprises the following components calculated by mass percentage: 40% recombinant humanized collagen, 35% probiotic composition and 25% glycogen.

[0089] Wherein, the preparation method of recombinant humanized collagen comprises:

[0090] (1) The nucleotide sequence of SEQ ID NO.6 was cloned into the prokaryotic expression vector pET22b (the plasmid was synthesized and constructed by Suzhou Hongxun Biotechnology Co., Ltd.). The plasmid vector pET22b carries a 6*His tag. The synthesized plasmid was transformed into E. coli BL21 competent cells.

[0091] SEQ ID NO.6 is the nucleotide sequence of SEQ ID NO.2, as follows:

[0092] GGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGT

[0093] (2) Pick a single recombinant Escherichia coli clone and transfer it to TB medium containing ampicillin resistance (50 ng / μL), and culture it overnight with shaking at 37 °C. The next day, inoculate the activated strain into 1 L of fresh TB medium for enlarged culture at a ratio of 1:100. When the OD600 reaches 0.6 at 37 °C with shaking at 180 rpm, cool down to 18 °C, add isopropyl β-D-thiogalactopyranoside (IPTG), and the final concentration of IPTG is 1 mM. Continue to induce expression for 18 h under the same conditions, and then collect the bacterial solution. Centrifuge at 5000 rpm for 30 min at 4 °C to collect the bacterial cells, resuspend and wash the cells with 50 mL of ultrapure water ddH2O, and then centrifuge again to collect the bacterial cells. Resuspend the collected bacterial cells with 50 mL of ddH2O, and then use a high-pressure cell disruptor to lyse the bacteria at a pressure of 800 Pa for 10 min at 4 °C. Centrifuge the lysed solution at 10000 rpm for 1 h at 4 °C, discard the precipitate, and collect the supernatant for subsequent purification.

[0094] (3) After the supernatant is adsorbed by a nickel ion column, it is successively washed with an aqueous solution of 20 mM PBS + 200 mM NaCl + 50 mM imidazole, pH = 7.4 and an aqueous solution of 20 mM PBS + 200 mM NaCl + 100 mM imidazole, pH = 7.4 to remove impurities, and then eluted with an aqueous solution of 20 mM PBS + 200 mM NaCl + 200 mM imidazole, pH = 7.4 and an aqueous solution of 20 mM PBS + 200 mM NaCl + 500 mM imidazole, pH = 7.4. The eluate is further desalted using a G25 gel column; subsequently, it is centrifuged at a centrifugal force of 10,000 g for 10 min, the supernatant is collected and concentrated using a 10 KD ultrafiltration tube (Millipore), centrifuged at 4,500 g, ultrafiltered to 500 μL at 4 °C, and the concentrate is washed three times with 1×PBS and centrifuged three times repeatedly to obtain a collagen PBS solution (pH = 6.4) with a mass percentage of 0.5%, which is stored at -80 °C for later use.

[0095] The gel electrophoresis results of the recombinant humanized collagen are as Figure 1 shown. An obvious specific band can be observed at around 20 kDa, which is basically consistent with the predicted molecular weight, indicating that the target protein is successfully expressed and secreted extracellularly, and the purity reaches over 90%. Among them, lane M: protein Marker.

[0096] (4) The stabilizer is dissolved in the above-mentioned collagen PBS solution to obtain a freeze-drying solution, and then freeze-dried to obtain a recombinant humanized collagen lyophilized powder; the stabilizer includes glycine, trehalose, mannitol, and Tween 80. The mass percentage of glycine in the freeze-drying solution is 1%, the mass percentage of trehalose in the freeze-drying solution is 2%, the mass percentage of mannitol in the freeze-drying solution is 2%, and the mass percentage of Tween 80 in the freeze-drying solution is 0.003%; the freeze-drying conditions include: pre-freezing at -20 °C for 8 h, and then vacuum freeze-drying at -40 °C for 36 h.

[0097] Comparative Example 2

[0098] The difference from Example 1 is that the recombinant humanized collagen composition for anti-HPV infection, calculated by mass percentage, includes the following components: probiotic composition 58%, glycogen 42%.

[0099] Comparative Example 3

[0100] The difference from Example 1 is that the recombinant humanized collagen composition for anti-HPV infection, calculated by mass percentage, includes the following components: recombinant humanized collagen fusion protein 61.5%, glycogen 38.5%.

[0101] Performance tests were conducted on the compositions prepared in the above examples and comparative examples:

[0102] Test Example 1 Adhesion Test

[0103] (1) In a laminar flow hood, dissolve the composition powder to be tested in MRS medium without glucose at 37 °C at a solid-liquid ratio of 1 mg: 1 mL. After the composition is completely dissolved, it is used as the sample solution to be tested. The sample solution to be tested is serially diluted and the viable cell count is performed to calculate the viable cell count P1 (CFU / g) per unit mass of the composition and the viable cell count P2 (CFU / mL) per unit volume of the sample solution to be tested.

[0104] (2) Transplant human vaginal epithelial cells VK2 / E6E7 into a 12-well plate and culture them using DMEM F12 medium. When the cell confluence reaches 80%, aspirate the medium and wash twice with PBS, then add the sample solution to be tested in step (1), 1 mL / well, and then incubate at 5% CO2 and 37 °C for 2 h and 4 h respectively.

[0105] (3) Carefully remove the culture supernatant, add PBS and rinse three times to remove non-adherent bacteria; add 0.2 mL / well of trypsin cell digestive solution and digest for 5 min to detach the cells from the well wall of the cell culture plate, and collect the solution for serial dilution and viable cell count.

[0106] The adhesion ability of probiotics to human vaginal epithelial cells was evaluated by adhesion ability and adhesion rate, and the calculation formulas are as follows:

[0107] Adhesion ability (CFU / cell) = number of bacteria adhered to cells (CFU) / number of epithelial cells in the well × 100%.

[0108] Adhesion rate (%) = number of bacteria adhered to cells (CFU) / total number of bacteria added to the well × 100%

[0109] The results are listed in Table 2.

[0110] Table 2

[0111]

[0112]

[0113] Comparing Examples 1 to 3, it can be seen that the more the amount of the recombinant humanized collagen fusion protein, the stronger the adhesion ability of the probiotic to epithelial cells, and the faster the adhesion speed. Comparing Examples 1, 4, and 5, it can be seen that the more the amount of the probiotic composition, the greater the number of viable bacteria per unit mass of the composition. In Comparative Example 1, recombinant humanized collagen was used instead of the fusion protein, and the adhesion ability of the probiotic to epithelial cells was greatly weakened; in Comparative Example 2, no collagen or fusion protein was added, and the probiotic had almost no adhesion ability to epithelial cells. In summary, the fusion protein can improve the adhesion ability of the probiotic to epithelial cells. The stronger the adhesion ability, the stronger the colonization ability of the probiotic, and it is easier to remain in the vaginal environment for reproduction, thereby playing the functions of inhibiting pathogenic bacteria, protecting the vaginal mucosa, and restoring the vaginal flora, achieving the curative effect of treating or preventing vaginal infections.

[0114] Test Example 2 Antibacterial Test

[0115] Preparation of the suspension of harmful bacteria: Gardnerella vaginalis, Escherichia coli, and Staphylococcus aureus were respectively activated on plate media, and the bacterial colonies were picked and transferred to normal saline to prepare a bacterial suspension, and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL; Candida albicans was activated by shaking flask YPD liquid culture for 16 h, and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL.

[0116] Antibacterial experiment: YPD medium, BHI medium supplemented with 5% defibrinated sheep blood, eosin methylene blue agar medium, and LB agar medium were added to the bacterial suspensions containing Candida albicans, Gardnerella vaginalis, Escherichia coli, and Staphylococcus aureus respectively, so that the number of viable harmful bacteria was 10 6 CFU / mL, and then quickly poured into the plate pre-placed with Oxford cups. After the medium cooled and solidified, the Oxford cups were taken out, and 200 μL of the test sample solution at 1 mg / mL (prepared in the same way as Test Example 1) was injected into each well. After culturing at 37 °C for 12 h, the diameter of the antibacterial zone was measured, and the results are shown in Table 3.

[0117] Table 3

[0118]

[0119]

[0120] Note: The diameter of the antibacterial zone includes the outer diameter of the Oxford cup, 7.80 mm.

[0121] As can be seen from the data in Table 3, the composition provided by the present invention has antibacterial activity against harmful vaginal bacteria. In Comparative Example 3, no probiotic was added, and almost no antibacterial activity was observed in the composition. In Comparative Example 1, recombinant human collagen fragments were used instead of the fusion protein, and the antibacterial activity decreased. The reason is that the fusion protein can better maintain the activity of probiotics and is beneficial to the proliferation of probiotics; in Comparative Example 2, no protein was added, so the antibacterial effect was worse than that of Comparative Example 1. Examples 1 to 3 had similar probiotic contents and similar antibacterial effects. In Example 5, due to the high probiotic content, the antibacterial effect was the best.

[0122] Test Example 3 Vaginal Mucosa Irritation Test

[0123] The vaginal mucosa irritation test was carried out in accordance with the "Disinfection Technology Specification" (Third Edition), "Disinfectant Toxicology Experiment Technology". Twelve healthy, adult female rabbits weighing 2 kg to 3 kg were randomly divided into 4 groups, with 3 rabbits in each group, corresponding to Example 1, Example 3, Example 5 and the blank control group respectively; once a day, vaginal administration was performed using a syringe, and the drug was 0.01 mL of the test sample with a concentration of 0.05 g / mL. The blank control group was administered an equal amount of normal saline. After continuous administration for 7 days, 24 hours after the last administration, the animals were sacrificed by air embolism, and the intact vaginal tissue was taken out by laparotomy. The vagina was longitudinally incised to observe manifestations such as congestion and edema. Then the vaginal tissue was fixed in 10% formalin solution for 24 hours, and tissues from 3 parts at both ends and the center of the vagina were selected for making slides. After HE staining, histopathological examination was carried out. The result evaluation was carried out according to the vaginal mucosa irritation reaction scoring standard in the "Disinfection Technology Specification" to determine the irritation reaction intensity of the vaginal mucosa irritation intensity classification.

[0124] Table 4

[0125]

[0126] As can be seen from Table 4, the vaginal mucosa irritation index of the composition of the present invention is less than or equal to 1, and the irritation reaction intensity is extremely mild irritation, all of which are within the specified range.

[0127] Application Example 1

[0128] A vaginal gel, calculated by mass percentage, includes the following components: 4% of the composition of Example 1, 10% of carbomer U20, 2% of sodium carboxymethylcellulose, 1% of glycerol, 2% of 1,3-propanediol, 2% of sodium hyaluronate, 1% of plant extract, 0.02% of lactic acid, and the balance is water and PBS buffer.

[0129] Among them, the plant extract, calculated by mass percentage, includes the following components: 20% green tea extract, 50% chamomile extract, 20% aloe extract, 10% tea tree oil from Australia.

[0130] A preparation method of a vaginal gel, comprising the following steps:

[0131] (1) Dissolve the composition of Example 1 with 1×PBS solution to prepare a 20% composition solution for later use.

[0132] (2) Dissolve the gel matrix, humectant, and thickener in water, stir evenly, cool down to room temperature, add the plant extract and stir evenly, then add lactic acid to adjust the pH value to 3.8, and finally slowly add the composition solution of step (1) to obtain the finished product.

[0133] Application Example 2

[0134] A vaginal gel, calculated by mass percentage, comprises the following components: 2% of the composition of Example 1, 10% of poloxamer 407; 0.5% of poloxamer 188, 0.2% of xanthan gum, 1% of sodium carboxymethylcellulose, 3% of glycerol, 0.5% of sodium hyaluronate, 2% of plant extract, 0.04% of lactic acid, and the balance is water and PBS buffer.

[0135] Among them, the plant extract, calculated by mass percentage, comprises the following components: 20% green tea extract, 50% chamomile extract, 20% aloe extract, and 10% tea tree oil from Australia.

[0136] The preparation method is the same as that of Application Example 1.

[0137] Application Example 3

[0138] A vaginal gel, calculated by mass percentage, comprises the following components: 6% of the composition of Example 1, 12% of poloxamer 407; 1% of poloxamer 188, 1% of sodium carboxymethylcellulose, 5% of glycerol, 3% of 1,3-propanediol, 2% of sodium hyaluronate, 1% of plant extract, 0.06% of lactic acid, and the balance is water and PBS buffer.

[0139] Among them, the plant extract, calculated by mass percentage, comprises the following components: 20% green tea extract, 50% chamomile extract, 20% aloe extract, and 10% tea tree oil from Australia.

[0140] The preparation method is the same as that of Application Example 1.

[0141] Application Comparative Example 1

[0142] The difference from Application Example 1 is that the composition of Comparative Example 1 is used instead of the composition of Example 1.

[0143] Application Comparative Example 2

[0144] The difference from Application Example 1 is that the composition of Comparative Example 2 is used instead of the composition of Example 1.

[0145] Application Comparative Example 3

[0146] It is different from Application Example 1 in that the composition of Comparative Example 3 is used instead of the composition of Example 1.

[0147] Perform the following performance tests on the vaginal gel prepared in the application example:

[0148] Test Example 5 Anti-HPV Infection

[0149] Experimental animals: SD rats, female, weighing 220 g - 260 g, 200 rats, randomly grouped, 40 rats in each group.

[0150] (1) Administer vaginal medication to the rats. Use a syringe to send the vaginal gels of Application Example 1 and Application Comparative Examples 1 - 3 deep into the vaginas of the rats. The dosage each time is 0.8 ± 0.05 mg, once a day, for one month, to obtain the rats to be infected; the blank control group is injected with an equal amount of normal saline.

[0151] (2) Prepare competent Gardnerella cells, perform transformation of the plasmid containing HPV18, and then infect the rats with the co-culture mixture of competent Gardnerella cells and Candida albicans. Among them, the volume and concentration of the HPV18 plasmid extract are 0.1 mL and 200 pmol / L, and the volume of the co-culture mixture of competent Gardnerella cells and Candida albicans is 0.1 mL;

[0152] (3) Randomly select half of the rats in each group and continue to administer vaginal medication according to step (1) on the sixth day of infection, and continue for two months;

[0153] (4) Adopt the TCT detection method to observe whether the cervical cells of the rats in each group have canceration or precancerous lesions (abnormalities) on the fifth day of infection and two months after infection, and perform molecular detection of the HPV virus. Statistically calculate the HPV infection rate (= number of infected rats / total number of rats × 100%) and the cervical cell canceration rate (= number of rats with abnormal cervical cells / total number of rats × 100%). The results are listed in Table 5.

[0154] Table 5

[0155]

[0156] It can be seen from the data in Table 5 that the vaginal gel provided in Application Example 1 of the present invention has a preventive and therapeutic effect on HPV. While in Application Comparative Example 1, pure recombinant humanized collagen is used instead of the fusion protein, in Application Comparative Example 2, the fusion protein is not added, and in Application Comparative Example 3, the probiotic composition is not added, resulting in poor prevention and treatment effects on HPV.

[0157] Test Example 6 Moisturizing Performance

[0158] The moisturizing property of the vaginal gel on human skin was determined using a skin moisture meter, and the specific steps are as follows:

[0159] Ten subjects were selected, and three 5×5 cm² experimental areas were demarcated on each of the two forearms, for a total of six, corresponding to six test samples; the tester sat still in a constant environment for 30 min, and then the mmv value of the skin without applying the vaginal gel was measured using a skin moisture meter. Then, 0.05 g of the test sample was evenly applied to the experimental area; the mmv values of the experimental area were detected at 30 min, 60 min, and 120 min after application, and the average value was calculated; the entire study was carried out in a laboratory with an indoor temperature of 22°C to 24°C and a humidity of 50% to 60%.

[0160] The results are listed in Table 6.

[0161] Table 6

[0162]

[0163] From the data in the table, it can be seen that the vaginal gel provided by the present invention has good moisturizing performance.

[0164] Test Example 7 Determination of Bacterial Viability

[0165] The application examples were serially diluted and viable bacteria were counted to obtain the number of viable bacteria contained in each unit of vaginal gel. The results are listed in Table 7.

[0166] Table 7

[0167] Sample Number of viable bacteria, CFU / g Application Example 1 <![CDATA[10 6 <!-- 13 -->]]> Application Example 2 <![CDATA[10 5 > Application Example 3 <![CDATA[10 6 > Application Comparative Example 1 <![CDATA[10 4 > Application Comparative Example 2 <![CDATA[10 4 >

[0168] From the data in the table, it can be seen that the probiotics have higher activity in the vaginal gel containing the recombinant humanized collagen fusion protein.

[0169] In summary, the present invention provides a recombinant humanized collagen composition for anti-HPV infection. The recombinant humanized collagen fusion protein in the composition can promote the adhesion and colonization of the probiotic composition on vaginal epithelial cells, has antibacterial activity against harmful bacteria, is less irritating, can quickly improve the vaginal microbial environment, and effectively enhance the resistance of the vagina to harmful bacteria and viruses.

[0170] The present invention provides a recombinant humanized collagen composition for anti-HPV infection. When the composition is applied to a vaginal gel, the probiotics therein can still maintain high activity, and it has preventive and therapeutic effects on HPV infection, as well as good moisturizing properties.

[0171] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant humanized collagen composition for resisting HPV infection, characterized in that: Calculated by mass percentage, it includes the following components: 30% to 45% of recombinant humanized collagen fusion protein, 30% to 40% of probiotics, and 20% to 35% of glycogen; the recombinant humanized collagen fusion protein includes a recombinant humanized collagen fragment and a human fibronectin fragment connected in sequence from the N-terminus to the C-terminus; the recombinant humanized collagen fragment and the human fibronectin fragment are connected by a linker; the amino acid sequence of the recombinant humanized collagen fragment is shown in SEQ ID NO.2; the amino acid sequence of the human fibronectin fragment is shown in SEQ ID NO.

3.

2. The recombinant humanized collagen composition for resisting HPV infection according to claim 1, characterized in that: The amino acid sequence of the recombinant humanized collagen fusion protein is shown in SEQ ID NO.

1.

3. The recombinant humanized collagen composition for resisting HPV infection according to claim 2, characterized in that: The recombinant humanized collagen fusion protein also includes a HIS tag fragment.

4. The recombinant humanized collagen composition for resisting HPV infection according to any one of claims 1 to 3, characterized in that: The probiotics include at least two of Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus rhamnosus and Lactobacillus reuteri; Preferably, the probiotics are a mixture of Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus rhamnosus and Lactobacillus reuteri in a live bacteria count ratio of 1:0.2:0.4:0.2; Preferably, the total number of viable bacteria in the recombinant humanized collagen composition for resisting HPV infection is 10 6 ~10 9 CFU / g.

5. The recombinant humanized collagen composition for resisting HPV infection according to any one of claims 1 to 4, characterized in that: The glycogen includes at least one of β-glucan, isomaltooligosaccharide, inulin, fructooligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, stachyose, manno-oligosaccharide and arabino-oligosaccharide.

6. The recombinant humanized collagen composition for resisting HPV infection according to any one of claims 1 to 4, characterized in that: The method for preparing the recombinant humanized collagen fusion protein comprises the following steps: An expression vector containing a nucleic acid molecule encoding the recombinant humanized collagen fusion protein according to any one of claims 1 to 4 is constructed, the expression vector is transferred into a recipient cell, the recipient cell is cultured, expression is induced, the cells are disrupted, and purification is performed to obtain the recombinant humanized collagen fusion protein.

7. The recombinant humanized collagen composition for resisting HPV infection according to claim 6, characterized in that: The method for preparing the recombinant humanized collagen fusion protein also includes mixing the recombinant humanized collagen fusion protein with a stabilizer and freeze-drying the mixture.

8. The method for preparing the recombinant humanized collagen composition for resisting HPV infection according to any one of claims 1 to 7, characterized in that: The steps include: (1) obtaining solid recombinant humanized collagen fusion protein, solid probiotics and glycogen; preferably, the solid recombinant humanized collagen fusion protein is a lyophilized powder of the recombinant humanized collagen fusion protein; and the solid probiotics are lyophilized probiotic powder; (2) Mixing the solid recombinant humanized collagen fusion protein, the solid probiotics, and the glycogen.

9. A preparation comprising the recombinant humanized collagen composition for resisting HPV infection according to any one of claims 1 to 7, characterized in that: The preparations are capsules, gels, tablets, suppositories, creams or effervescent tablets.

10. A vaginal gel, characterized in that: Calculated by mass percentage, the composition comprises the following components: 2% to 6% of the recombinant humanized collagen composition for resisting HPV infection as claimed in any one of claims 1 to 7, 8% to 15% of the gel matrix, 1% to 3% of the thickener, 3% to 10% of the moisturizer, 1% to 2% of the plant extract, 0% to 1% of the pH adjuster, and the balance is water and PBS buffer.

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