A recombinant humanized collagen composition against HPV infection and a preparation method thereof

Abstract

The application discloses a recombinant humanized collagen composition for resisting HPV infection and a preparation method thereof. The composition comprises the following components in percentage by mass: 30-45% of a recombinant humanized collagen fusion protein, 30-40% of probiotics and 20-35% of glycogen. The recombinant humanized collagen fusion protein comprises, from N-terminal to C-terminal, a recombinant humanized collagen fragment and a human fibronectin fragment connected in sequence. The recombinant humanized collagen fragment and the human fibronectin fragment are connected by a linker. The recombinant humanized collagen fusion protein in the composition can promote the adhesion and colonization of the probiotic composition to the 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 application relates to the technical field of biological medicine, in particular to a recombinant humanized collagen composition for resisting HPV infection and a preparation method thereof. BACKGROUND

[0002] HPV is a DNA virus that can exist in two forms, free or integrated, and achieve persistent infection by replication and transcription in host cells, causing cell tumorigenesis. The persistence of HPV infection is influenced by the surrounding microenvironment. The local microenvironment of the cervix plays an important role in maintaining tissue homeostasis, resisting pathogens, and participating in immune response, and can affect the natural history of HPV infection. Vaginal microbiota (VMB) is an important component of the cervical microenvironment, and its specific characteristics are closely related to gynecological diseases and reproductive health. The composition of VMB can affect the local immune response, and 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 HPV infection of the reproductive tract has attracted more and more attention from scholars. Researchers found that there were significant differences in vaginal microecology between HPV-negative normal women and HPV-positive women through high-throughput sequencing of vaginal secretions. HPV-positive women mainly showed a high degree of diversity in vaginal microorganisms, and a decrease in lactobacillus and an increase in Gardnerella vaginalis. In addition, a large number of literatures have proved that bacterial vaginosis (BV) is positively correlated with the incidence, prevalence and persistence of HPV infection. BV is actually a vaginal flora imbalance, mainly characterized by a decrease in lactobacillus that secretes H2O2 in the vagina, and a large number of Gardnerella and anaerobes. Under normal circumstances, lactobacillus prevents the colonization of bacteria species related to bacterial vaginosis by maintaining low pH and producing bacteriocins, which is important for maintaining the barrier function of the cervical epithelium that inhibits HPV from entering the basal keratinocytes. When BV-related strict anaerobes can colonize, they will produce enzymes and metabolites that can damage this barrier, providing a good environment for HPV infection and invasion. HPV enters, especially high-risk HPV fragments and host gene integration, and finally undergoes tumor transformation. Therefore, HPV infection is closely related to the disorder of the local microecological environment of the vagina, especially the decrease in lactobacillus.

[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 treatment of vaginal administration of probiotics is long, and the reason is that probiotics do not quickly and effectively colonize the surface of vaginal mucosal epithelial cells, resulting in insufficient drug efficacy and a large amount of effective components being excreted outside the body, causing waste.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The present application provides a recombinant humanized collagen composition against HPV infection and a preparation method thereof. The composition comprises a recombinant humanized collagen fusion protein, glycogen and a probiotic composition. The recombinant humanized collagen fusion protein can improve the adhesion of beneficial bacteria to epithelial cells, promote the reproduction of the beneficial bacteria, 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 application is implemented as follows:

[0007] In a first aspect, the present application provides a recombinant humanized collagen composition against HPV infection, comprising the following components in terms of mass percentage: 30-45% of a recombinant humanized collagen fusion protein, 30-40% of probiotics, and 20-35% of glycogen; the recombinant humanized collagen fusion protein comprises, from N-terminal to C-terminal, a recombinant humanized collagen fragment and a human fibronectin fragment connected in sequence; the recombinant humanized collagen fragment and the human fibronectin fragment are connected using a linker; the amino acid sequence of the recombinant humanized collagen fragment is shown in SEQ ID NO. 2; and the amino acid sequence of the human fibronectin fragment is shown in SEQ ID NO. 3.

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

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

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

[0011] SEQ ID NO. 1:

[0012] GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGGGGGSVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT

[0013] SEQ ID NO. 1:

[0014] GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR

[0015] SEQ ID NO. 2:

[0016] VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT

[0017] In the present application, SEQ ID NO. 2 is a triple repeat sequence of SEQ ID NO. 4, and SEQ ID NO. 4 is obtained by screening and optimization from the sequence of human natural type III collagen COL-III. The sequence of the human collagen III is NCBI reference sequence: KAI4037253.1, see https: / / www.ncbi.nlm.nih.gov / protein / KAI4037253.1. The applicant found through a large amount of research that the above-mentioned sequence has strong water solubility, high recombinant expression yield, simple purification process, and achieves better biological activity than other sequences of commercial collagen proteins.

[0018] SEQ ID NO. 4:

[0019] GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR

[0020] SEQ ID NO. 3 is a fragment of human fibronectin (FN), which is a glycoprotein of extracellular matrix. Fibronectin can bind extracellular matrix components such as collagen, fibrin and heparin sulfate proteoglycan (such as multiple ligand glycan) and participate in cell adhesion, cell movement, opsonization, wound healing and maintenance of cell shape. In addition, fibronectin can bind to integrin αvβ3 on the cell surface to enhance cell adhesion. The selected SEQ ID NO. 3 fragment has good fusion effect with SEQ ID NO. 2, i.e. 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 shown in SEQ ID NO. 5.

[0022] SEQ ID NO. 5

[0023] GGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGTGGTGGTGGTGGTTCTGTCTCAGATGTACCCAGGGACCTAGAAGTTGTCGCAGCGACCCCGACCAGCTTGCTGATTTCCTGGGATGCGCCTGCGGTTACCGTTCGTTATTACCGTATTACCTATGGTGAGACAGGCGGAAACAGCCCGGTTCAAGAATTCACCGTGCCAGGTTCGAAAAGCACCGCAACGATTTCTGGCCTGAAACCGGGCGTCGATTATACCATCACCGTTTACGCAGTTACGGGTAGAGGCGACAGCCCAGCGTCTTCCAAACCGATTAGTATTAACTACCGCACC

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

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

[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 beta-glucan, isomalto-oligosaccharide, inulin, fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, stachyose, manno-oligosaccharide, and arabo-oligosaccharide.

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

[0029] The expression vector containing the nucleic acid molecule encoding the recombinant humanized collagen fusion protein in any of the above embodiments is constructed, the expression vector is transformed into a recipient cell, the recipient cell is cultured, expression is induced, the bacterial cells are broken, and purification is performed 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, and specifically includes: adding a stabilizer to a PBS solution (pH = 6.4-7.4) of the recombinant humanized collagen fusion protein at a mass percentage of 0.01%-0.5% to obtain a freeze-dried liquid, and then freeze-drying to obtain a freeze-dried powder of the recombinant humanized collagen fusion protein.

[0031] In some embodiments, the stabilizer includes glycine, trehalose, mannitol, and Tween 80, the glycine accounts for 1%-2% of the mass percentage of the freeze-dried liquid, the trehalose accounts for 0.5%-1.5% of the mass percentage of the freeze-dried liquid, the mannitol accounts for 2%-4% of the mass percentage of the freeze-dried liquid, and the Tween 80 accounts for 0.001%-0.004% of the mass percentage of the freeze-dried liquid.

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

[0033] In a second aspect, the present application provides a method for preparing the recombinant humanized collagen composition against HPV infection according to any of the above embodiments, characterized in that it comprises the following steps:

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

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

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

[0037] In a fourth aspect, the present application provides a vaginal gel comprising the following components in percentage by mass: 2-6% of the recombinant humanized collagen composition, 8-15% of the gel base, 1-3% of the thickening agent, 3-10% of the humectant, 1-2% of the plant extract, 0-1% of the pH adjuster, and the balance of water and PBS buffer.

[0038] In some embodiments, the gel base 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 salt, hydroxyethyl cellulose, hydroxypropyl cellulose.

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

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

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

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

[0044] The present application provides a recombinant humanized collagen composition against HPV infection, which can promote the adhesion and colonization of probiotic composition on vaginal epithelial cells, rapidly improve the microbial environment of the vagina, and effectively enhance the resistance of the vagina to harmful bacteria and viruses.

[0045] The present application provides a recombinant humanized collagen composition against HPV infection, which has a high activity of probiotics and a good moisturizing property when applied to a vaginal gel, and has a preventive and therapeutic effect on HPV infection. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0047] Figure 1 Electrophoretogram of the recombinant humanized collagen fusion protein prepared in Example 1 and the recombinant humanized collagen fragment prepared in Comparative Example 1. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

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

[0050] Reference to "embodiments" herein means that the specific features, structures or properties described in connection with the embodiments can be included in at least one embodiment of the present application. The appearance of this phrase at various places in the specification does not necessarily refer to the same embodiment, nor is it independent or alternative to other embodiments.

[0051] In the embodiments of the present application, the term "or / and" is only a description of the association relationship between the associated objects, which means that there can be three relationships, for example, A or / and B, which can mean that A exists alone, A and B exist together, and B exists alone.

[0052] In addition, the character " / " in this paper generally represents that the front and rear associated objects are in an "or" relationship.

[0053] In the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similarly, "multiple groups" means more than two groups (including two groups), and "multiple layers" means more than two layers (including two layers), unless otherwise specified and limited.

[0054] In the embodiments of the present application, the meaning of “at least one” is one or more than one.

[0055] In the embodiments of the present application, the directions or positional relationships indicated by the technical terms “length”, “width”, “thickness”, “upper”, “lower”, “front”, “rear”, “left”, “right”, “vertical”, “horizontal” and the like are the directions or positional relationships shown in the drawings, which are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed in a particular orientation, etc., and should not be understood as a limitation on the embodiments of the present application. Those skilled in the art can understand the specific meanings of the above-mentioned 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 part of the specific embodiments.

[0057] Table 1

[0058]

[0059] The features and performances of the present application are further described in detail below in combination with the embodiments.

[0060] Embodiment 1

[0061] A recombinant humanized collagen composition against HPV infection, comprising the following components by mass percentage: recombinant humanized collagen fusion protein 40%, probiotic composition 35%, glycogen 25%.

[0062] The preparation method of the recombinant humanized collagen fusion protein comprises:

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

[0064] (2) Positive recombinants were screened by ampicillin-resistant plates, and then the strain with correct sequencing was transferred to TB medium containing ampicillin resistance (50 ng / μL) and cultured at 37°C overnight. The next day, the activated strain was inoculated into 1 L of fresh TB medium at a ratio of 1:100, and cultured at 37°C with 180 rpm shaking until OD600=0.6. Then, the temperature was reduced to 18°C, and isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM. The induction was continued under the same conditions for 18 h, and then the bacterial solution was collected. The bacterial solution was centrifuged at 5000 rpm at 4°C for 30 min, and then resuspended and washed with 50 mL of ultrapure water ddH2O. The bacterial solution was resuspended with 50 mL of ddH2O, and then the bacteria were broken using a high-pressure disrupter at a pressure of 800 Pa at 4°C for 10 min. The broken solution was centrifuged at 10000 rpm at 4°C for 1 h, and the precipitate was discarded. The supernatant was collected for subsequent purification.

[0065] (3) The supernatant was adsorbed by a nickel ion column, and then washed with 20 mM PBS+200 mM NaCl+50 mM imidazole, pH=7.4 aqueous solution, 20 mM PBS+200 mM NaCl+100 mM imidazole, pH=7.4 aqueous solution, 20 mM PBS+200 mM NaCl+200 mM imidazole, pH=7.4 aqueous solution, and 20 mM PBS+200 mM NaCl+500 mM imidazole, pH=7.4 aqueous solution, respectively. The eluate was further desalted using a G25 gel column. Then, the solution was centrifuged at 10000 g for 10 min, and the supernatant was collected and concentrated using a 10 KD ultrafiltration tube (Millipore). The concentrated solution was washed with 1xPBS three times and centrifuged three times at 4500 g at 4°C to obtain a PBS solution (pH=6.4) of the fusion protein with a mass percentage of 0.5%, which was stored at -80°C for later use.

[0066] The gel electrophoresis result of the recombinant humanized collagen fusion protein is shown in Figure 1 As shown in the figure, a specific band of about 31 kDa was observed, which was basically consistent with the predicted molecular weight, indicating that the target protein was successfully expressed and secreted into the extracellular space, and the purity was more than 90%. Lane M: protein marker.

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

[0068] The probiotic composition is a mixture of Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus rhamnosus and Lactobacillus reuteri mixed in a ratio of 1:0.2:0.4:0.2 by number of viable bacteria.

[0069] The glycogen is a mixture of β-glucan, inulin and isomaltooligosaccharide mixed in a mass ratio of 1:1:0.3.

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

[0071] The freeze-dried powder of the recombinant humanized collagen fusion protein, the freeze-dried powder of the probiotic bacteria and the glycogen are mixed according to the formula.

[0072] Example 2

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

[0074] Example 3

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

[0076] Example 4

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

[0078] The recombinant humanized collagen fusion protein is the same as that in Example 1.

[0079] The probiotic composition is a mixture of Lactobacillus crispatus, Lactobacillus rhamnosus and Lactobacillus reuteri mixed in a ratio of 1:0.4:0.2 by number of viable bacteria.

[0080] wherein the glycogen is a mixture of beta-glucan and isomalto-oligosaccharide at a mass ratio of 1:1.

[0081] Example 5

[0082] A recombinant humanized collagen composition against HPV infection, comprising the following components by mass percentage: recombinant humanized collagen fusion protein 30%, probiotic composition 40%, glycogen 30%.

[0083] wherein the recombinant humanized collagen fusion protein is the same as that of Example 1;

[0084] wherein the probiotic composition is a mixture of Lactobacillus crispatus and Pediococcus acidilactici at a viable bacterial count ratio of 1:0.2.

[0085] wherein the glycogen is isomalto-oligosaccharide.

[0086] Comparative Example 1

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

[0088] A recombinant humanized collagen composition against HPV infection, comprising the following components by mass percentage: recombinant humanized collagen 40%, probiotic composition 35%, glycogen 25%.

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

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

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

[0092] GGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCA GGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCTGGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGAT GGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGTGGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACCGGGTCAGGCAGGTCCATCT GGTCCACCGGGTCCGCCGGGTGCTATTGGTCCTTCCGGCCCGGCTGGTAAAGATGGCGAATCTGGCCGCCCTGGTCGTCCGGGCGAACGTGGTCTGCCGGGTCCGCCAGGTATCAAAGGTCCGGCGGGCATCCCGGGTTTCCCTGGTATGAAAGGCCATCGT

[0093] (2) Select a single clone of recombinant *E. coli* and transfer it to TB medium containing ampicillin (50 ng / μL). Incubate overnight at 37°C with shaking. The next day, inoculate the activated bacterial culture at a ratio of 1:100 into 1 L of fresh TB medium for expansion culture. Incubate at 37°C with shaking at 180 rpm until OD600 = 0.6, then cool to 18°C ​​and add isopropyl thiogalactoside (IPTG) to a final concentration of 1 mM. Continue induction under the same conditions for 18 h and then collect the bacterial culture. Centrifuge at 5000 rpm at 4°C for 30 min and collect the bacterial cells. Resuspend the cells in 50 mL of ultrapure water (ddH2O), wash, and centrifuge again to collect the bacterial cells. Resuspend the collected bacterial cells in 50 mL of ddH2O, and then use a high-pressure homogenizer to disrupt the bacteria at a pressure of 800 Pa at 4°C for 10 min. Centrifuge the disrupted solution at 10000 rpm at 4°C for 1 h, discard the precipitate, and collect the supernatant for subsequent purification.

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

[0095] The gel electrophoresis result of the recombinant humanized collagen protein is shown in Figure 1 It can be observed that there is a specific band of about 20 kDa, which is basically consistent with the predicted molecular weight, indicating that the target protein is successfully expressed and secreted into the extracellular, and the purity is more than 90%, wherein lane M: protein marker.

[0096] (4) The stabilizer is dissolved in the above collagen protein PBS solution to obtain a freeze-dried liquid, and then freeze-dried to obtain a recombinant humanized collagen protein freeze-dried powder; the stabilizer includes glycine, trehalose, mannitol and Tween 80, the mass percentage of glycine in the freeze-dried liquid is 1%, the mass percentage of trehalose in the freeze-dried liquid is 2%, the mass percentage of mannitol in the freeze-dried liquid is 2%, and the mass percentage of Tween 80 in the freeze-dried liquid 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 protein composition against HPV infection comprises, by mass percentage, the following components: probiotic composition 58%, glycogen 42%.

[0099] Comparative Example 3

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

[0101] The compositions prepared in the above examples and comparative examples were subjected to performance tests.

[0102] Example 1 Adhesion Test

[0103] (1) In a super-clean bench, the composition powder to be tested was dissolved in 37°C MRS medium without glucose at a solid-liquid ratio of 1 mg: 1 mL. After the composition was completely dissolved, the sample liquid to be tested was subjected to gradient dilution and viable cell counting, and the number of viable cells per unit mass of the composition P1 (CFU / g) and the number of viable cells per unit volume of the sample liquid to be tested P2 (CFU / mL) were calculated.

[0104] (2) Human vaginal epithelial cells VK2 / E6E7 cells were transplanted into a 12-well plate and cultured with DMEM F12 medium. When the cell plating rate reached 80%, the culture medium was aspirated and rinsed with PBS twice, then the sample liquid to be tested in step (1) was added, 1 mL / well, and then incubated at 37°C in 5% CO2 for 2 h and 4 h, respectively.

[0105] (3) The culture supernatant was carefully removed and rinsed with PBS three times to remove unadhered bacteria; trypsin cell digestion solution 0.2 mL / well was added and digested for 5 min to detach the cells from the cell culture plate well wall, and the solution was collected for gradient dilution and viable cell counting.

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

[0107] Adhesion capacity (CFU / cell) = number of bacteria adhering to cells (CFU) / number of epithelial cells in well x 100%.

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

[0109] The results are shown in Table 2.

[0110] Table 2

[0111]

[0112]

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

[0114] Bacteriostatic test of Test Example 2

[0115] Preparation of harmful bacteria suspension: Gardnerella vaginalis, Escherichia coli and Staphylococcus aureus were activated on plate medium, and bacterial lawns were picked into physiological saline to prepare a bacterial suspension, and the concentration of the bacterial solution was adjusted to 10 8 CFU / mL; Candida albicans was activated by liquid YPD culture for 16 h, and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL.

[0116] Bacteriostatic experiment: YPD medium, BHI medium containing 5% defibrillated 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 viable bacteria of the harmful bacteria were 10 6 CFU / mL, and then quickly poured into a plate with an Oxford cup in advance. After the medium cooled and solidified, the Oxford cup was taken out, 200 μL of 1 mg / mL sample solution to be tested (prepared in the same way as in Test Example 1) was injected into each well, and after 12 h of culture at 37°C, the diameter of the bacteriostatic ring was measured, and the results are shown in Table 3.

[0117] Table 3

[0118]

[0119]

[0120] Note: The diameter of the bacteriostatic ring includes the outer diameter of the Oxford cup of 7.80 mm.

[0121] From the data in Table 3, it can be seen that the composition provided by the application has bacteriostatic activity on harmful bacteria in the vagina. Comparative Example 3 does not add probiotics, and the composition almost does not have bacteriostatic activity. Comparative Example 1 uses a recombinant human collagen fragment instead of a fusion protein, and the bacteriostatic activity decreases. The reason is that the fusion protein can better maintain the activity of probiotics and is beneficial to the proliferation of probiotics. Comparative Example 2 does not add protein, and therefore the bacteriostatic effect is poorer than that of Comparative Example 1. Examples 1-3 have similar probiotic contents and similar bacteriostatic effects. Example 5 has the highest probiotic content, and therefore has the best bacteriostatic effect.

[0122] Test Example 3: Vaginal mucosa irritation test

[0123] The vaginal mucosa irritation test was performed according to the “Disinfectant Toxicology Experiment Technology” in the “Disinfection Technical Specification” (third edition). Twelve healthy, adult, and 2-3 kg female rabbits were randomly divided into four groups, three rabbits in each group, corresponding to Examples 1, 3, 5, and the blank control group, respectively. The rabbits were given a vaginal dose of 0.01 mL of the test sample with a concentration of 0.05 g / mL once a day. The blank control group was given an equal amount of normal saline. The rabbits were continuously given the dose for 7 days. Twenty-four hours after the last dose, the animals were sacrificed by air embolism, and the complete vaginal tissue was removed by abdominal incision. The tissue was observed for signs of congestion and edema. Then, the vaginal tissue was fixed in 10% formalin solution for 24 hours. Tissue sections were prepared from the tissue at the two ends and the central part of the vagina. After HE staining, histopathological examination was performed. The results were evaluated according to the “Disinfection Technical Specification” for the vaginal mucosa irritation reaction scoring standard to determine the irritation reaction intensity.

[0124] Table 4

[0125]

[0126] As can be seen from Table 4, the composition of the application has a rabbit vaginal mucosa irritation index of less than or equal to 1, and the irritation reaction intensity is extremely slight irritation, which all meet the specified range.

[0127] Application Example 1

[0128] A vaginal gel, by mass percentage, comprises the following components: 4% of the composition of Example 1, 10% of carbomer U2, 2% of sodium hydroxymethyl cellulose, 1% of glycerol, 2% of 1,3-propanediol, 2% of sodium hyaluronate, 1% of plant extract, 0.02% of lactic acid, and the balance of water and PBS buffer.

[0129] The plant extract comprises the following components by mass percentage: 20% green tea extract, 50% chamomile extract, 20% aloe vera extract, and 10% tea tree oil from Australia.

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

[0131] (1) A 20% composition solution is prepared by dissolving the composition of Example 1 with 1x PBS solution and is ready for use.

[0132] (2) The gel matrix, humectant, thickening agent are dissolved in water, stirred uniformly, cooled to room temperature, then the plant extract is added and stirred uniformly, then lactic acid is added to adjust the pH value to 3.8, and finally the composition solution of step (1) is slowly added to obtain the finished product.

[0133] Application Example 2

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

[0135] The plant extract comprises the following components by mass percentage: 20% of green tea extract, 50% of chamomile extract, 20% of aloe extract, and 10% of tea tree oil.

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

[0137] Application Example 3

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

[0139] The plant extract comprises the following components by mass percentage: 20% of green tea extract, 50% of chamomile extract, 20% of aloe extract, and 10% of tea tree oil.

[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] The difference from application example 1 is that the composition of comparative example 3 is used instead of the composition of example 1.

[0147] The vaginal gel prepared in the application example is subjected to the following performance tests:

[0148] Test Example 5 Anti-HPV infection

[0149] Experimental animals: SD rats, female, body weight 220g-260g, 200, randomly divided into groups, 40 in each group.

[0150] (1) The rats are subjected to vaginal administration, and the vaginal gels of application example 1 and application comparative examples 1-3 are sent into the deep part of the rat vagina with a syringe, the amount used each time is 0.8±0.05 mg, once a day, for one month, to obtain the infected rats; the blank control group is injected with the same amount of normal saline.

[0151] (2) The Gardneri bacteria competent cells are prepared, the HPV18-containing plasmid is transformed, and then the rats are infected with the co-culture mixture of Gardneri bacteria competent 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 Gardneri bacteria competent cells and Candida albicans is 0.1 mL;

[0152] (3) Half of the number of rats in each group are randomly selected on the sixth day of infection to continue vaginal administration according to step (1), and for two months;

[0153] (4) The TCT detection method is used to observe whether the cervical cells of the rats in each group are cancerous or precancerous (abnormal) on the fifth day of infection and two months after infection, and the molecular detection of HPV virus is carried out, the HPV infection rate (= the number of infected rats / the total number of rats x 100%) and the cervical cell canceration rate (= the number of abnormal cervical cells / the total number of rats x 100%) are calculated, and the results are shown in Table 5.

[0154] Table 5

[0155]

[0156] From the data in Table 5, it can be seen that the vaginal gel provided by application example 1 has a preventive and therapeutic effect on HPV. The application comparative example 1 uses pure recombinant humanized collagen instead of the fusion protein, the application comparative example 2 does not add the fusion protein, and the application comparative example 3 does not add the probiotic composition, resulting in poor prevention and treatment of HPV.

[0157] Test Example 6 Moisturizing performance

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

[0159] Ten subjects are selected, and two forearms are respectively marked with 3 5*5 cm2 experimental areas, a total of 6, corresponding to 6 samples to be tested; the tester sits still for 30 min in a constant environment, and then the mmv value of the skin without coating the vaginal gel is determined by using a skin moisture meter, then 0.05 g of the sample to be tested is evenly coated on the experimental area; the mmv value of the experimental area is detected at 30 min, 60 min and 120 min after coating, and the average value is obtained; the whole study is carried out in a laboratory with indoor temperature of 22-24 DEG C and humidity of 50-60%.

[0160] The results are shown 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 application has good moisturizing property.

[0164] Test Example 7 Bacterial activity determination

[0165] Each application example is gradiently diluted and live bacteria are counted to obtain the number of live bacteria contained in unit vaginal gel, and the results are shown in Table 7.

[0166] Table 7

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

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

[0169] In summary, the application provides a recombinant humanized collagen composition for resisting HPV infection, the recombinant humanized collagen fusion protein in the composition can promote the adhesion and colonization of the probiotic composition on the vaginal epithelial cells, has bacteriostatic activity on harmful bacteria, has weak stimulation, can quickly improve the microbial environment of the vagina, and effectively enhances the resistance of the vagina to harmful bacteria and viruses.

[0170] The application provides a recombinant humanized collagen composition for resisting HPV infection, the probiotics in the composition can still maintain high activity when the composition is applied to the vaginal gel, and have the effects of preventing and treating HPV infection, and have good moisturizing property.

[0171] The above merely describes the preferred embodiments of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant humanized collagen composition against HPV infection, characterized in that, According to the mass percentage, the composition comprises the following components: 30%-45% of recombinant humanized collagen fusion protein, 30%-40% of probiotics, and 20-35% of glycogen; the recombinant humanized collagen fusion protein comprises, from N-terminal to C-terminal, a recombinant humanized collagen fragment and a human fibronectin fragment connected in sequence; 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; the amino acid sequence of the recombinant humanized collagen fusion protein is shown in SEQ ID NO. 1; the probiotics comprise at least two of lactobacillus crispatus, pediococcus acidilactici, lactobacillus rhamnosus, and lactobacillus reuteri; and the glycogen comprises at least one of beta-glucan, isomalto-oligosaccharide, inulin, fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, stachyose, manno-oligosaccharide, and arabo-oligosaccharide.

2. The recombinant humanized collagen composition for anti-HPV infection according to claim 1, characterized in that, The recombinant humanized collagen fusion protein further comprises a HIS tag fragment.

3. The recombinant humanized collagen composition for combating HPV infection according to any one of claims 1 to 2, characterized in that, The probiotics are mixed bacteria of lactobacillus crispatus, pediococcus acidilactici, lactobacillus rhamnosus, and lactobacillus reuteri in a ratio of 1:0.2:0.4:0.

2.

4. The recombinant humanized collagen composition for anti-HPV infection according to any one of claims 1 to 2, characterized in that, The total number of viable bacteria in the recombinant humanized collagen composition against HPV infection is 10 6 ~10 9 CFU / g.

5. The recombinant humanized collagen composition for use against HPV infection according to claim 3, characterized in that, The total number of viable bacteria in the recombinant humanized collagen composition against HPV infection is 10 6 ~10 9 CFU / g.

6. The recombinant humanized collagen composition for anti-HPV infection according to any one of claims 1 to 2, characterized in that, The preparation method of the recombinant humanized collagen fusion protein comprises the following steps: constructing an expression vector containing a nucleic acid molecule encoding the recombinant humanized collagen fusion protein according to any one of claims 1-2, transforming the expression vector into a recipient cell, culturing the recipient cell, inducing expression, breaking the bacterial cells, and purifying to obtain the recombinant humanized collagen fusion protein.

7. The recombinant humanized collagen composition for use against HPV infection according to claim 6, characterized in that, The preparation method of the recombinant humanized collagen fusion protein further comprises mixing the recombinant humanized collagen fusion protein with a stabilizer and freeze-drying.

8. The method of claim 1 to 7, wherein the method of preparing the recombinant humanized collagen composition for anti-HPV infection is characterized by, The method comprises the following steps: (1) obtaining solid recombinant humanized collagen fusion protein, solid probiotics, and glycogen; (2) mixing the solid recombinant humanized collagen fusion protein, the solid probiotics, and the glycogen.

9. The method for preparing the recombinant humanized collagen composition for anti-HPV infection according to claim 8, characterized in that, The solid recombinant humanized collagen fusion protein is a freeze-dried powder of the recombinant humanized collagen fusion protein; and the solid probiotics are a freeze-dried probiotic powder.

10. A preparation comprising the recombinant humanized collagen composition for preventing HPV infection according to any one of claims 1 to 7, characterized in that, The preparation is a capsule, a gel, a tablet, a suppository, a cream, or an effervescent tablet.

11. A vaginal gel characterized in that, According to the mass percentage, the composition comprises the following components: 2%-6% of the anti-HPV infection recombinant humanized collagen composition according to any one of claims 1-7, 8%-15% of a gel base, 1%-3% of a thickening agent, 3%-10% of a humectant, 1%-2% of a plant extract, 0-1% of a pH adjuster, and the balance of water and PBS buffer.

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