CN109575126B - Polypeptides, method for the production and use thereof - Google Patents

Polypeptides, method for the production and use thereof Download PDF

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CN109575126B
CN109575126B CN201910111553.7A CN201910111553A CN109575126B CN 109575126 B CN109575126 B CN 109575126B CN 201910111553 A CN201910111553 A CN 201910111553A CN 109575126 B CN109575126 B CN 109575126B
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杨霞
陆路
姜世勃
花晨
刘泽众
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Shanxi Jin Bo Biomedics Inc
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Abstract

The present invention relates to polypeptides, methods for their production and uses thereof. The polypeptide comprises an N-terminal region and a C-terminal region, wherein the N-terminal region comprises an N-terminal sequence of at least 48 consecutive amino acid residues of SEQ ID No. 13; wherein the C-terminal region comprises the sequence GPPGPCCGGG shown as SEQ ID No.14 or comprises the amino acid sequence shown as SEQ ID No. 18. The polypeptide of the invention has obviously better adhesion effect than the commercialized human collagen. The polypeptide of the invention has anti-HPV property. The invention provides a new method for further optimizing and treating HPV infection diseases such as cervical cancer and the like.

Description

Polypeptides, method for the production and use thereof
The present application claims priority of chinese patent application No. 201810736734.4 entitled "polypeptide, method of producing the same, and use thereof", filed on 6.7.2018.
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to a polypeptide, a production method and application thereof.
Background
Cervical cancer and papilloma virus
Cervical cancer ranks fourth in the incidence of female cancer worldwide and has been a focus of attention in the international medical field. The cancer is confirmed to be caused by Human Papillomavirus (HPV), which has more than 200 subtypes and can be divided into a low-risk type and a high-risk type according to the carcinogenicity of the HPV, wherein the low-risk type has high prevalence and represents HPV6, HPV11 and the like, and the HPV mainly causes benign genital warts and low cervical epithelial necrosis. The high-risk types comprise HPV16, HPV18, HPV52, HPV58 and the like, and can cause cervical cancer.
It was shown by WHO data that the disease caused death in 27 million people in 2012. Of which over 85% occurs in low and medium income countries. Cervical cancer is the second most common cancer in women living in less developed areas, with an estimated 44.5 million new cases in 2012, accounting for 84% of new cases worldwide. With the deepening of the sexual openness degree of China, the incidence rate and the onset age of cervical cancer are likely to have obvious rising and advancing trends.
The occurrence and development of cervical cancer are related to virus persistent infection, the current treatment of the cervical cancer is mainly to excise a focus through surgery, and the common treatment methods comprise: cyclic electrosurgery (LEEP), cold-knife conization, electroshock therapy, cryotherapy, and the like. However, due to the characteristics of HPV infection, the method such as operation cannot play a role in preventing virus infection, cannot completely eliminate virus, and has high recurrence rate. At present, three vaccines are marketed aiming at human papilloma virus, and the three vaccines can effectively prevent the infection of the popular HPV type and can prevent cervical cancer. There are still some limitations: for example, (1) vaccination is limited. The 9-valent vaccine in China has just been approved, and some people outside the age range are not suitable for the vaccine; (2) type limitation: the three vaccines that have been marketed prevent infection by 9 subtypes of human papillomavirus at the most, but have no preventive effect on infection by other HPV types. From the viewpoint of virus ecology, when major types 9 of HPV are suppressed, other types of HPV may become major epidemic strains; (3) the cost is high, the price is expensive, and the popularization of the vaccine in developing countries is limited; (4) the current vaccines are all preventive vaccines, and no clinical data support has treatment effect on patients infected with human papilloma virus. Therefore, there is an urgent need to develop products that can effectively prevent and control HPV viral infection.
Limited by the development of virology and the characteristics of human papillomavirus, the product is currently evaluated in the world in the absence of a live virus system of human papillomavirus, and an ideal and accepted animal model is absent, for the condition, the currently accepted in vitro model for researching the HPV product is an HPV pseudovirus model which consists of HPV L1, L2 protein and a reporter gene and is an in vitro model widely used by the academic circles at home and abroad for detecting the activity of anti-human papillomavirus drugs. The activity of the product can be visually evaluated by means of the model.
The invention provides a protein with significant anti-HPV activity, and provides a new method for further optimizing and treating HPV infection diseases such as cervical cancer and the like.
Collagen protein
Collagen is generally white, transparent and unbranched fibril, is a basic support of skin and bones, can account for 25% -35% of the total amount of protein, is mainly distributed in skin, blood vessels, bones, tendons, teeth, cartilage and the like of a human body, is a main matrix and a support of the tissues, protects and binds various tissues, and plays an important physiological function in a body. Therefore, the collagen can be widely applied to industries such as medicine, cosmetics and the like.
Collagen products currently on the market are all derived from animal tissues such as pigs, cattle, fish, etc. In terms of the amino acid composition of collagen: the similarity between the mammal pig and the mammal cow is 95 percent, and the similarity between the mammal pig and the mammal cow is 65 percent. Although the similarity of collagen of mammals such as pig and cow to human is high, it is still difficult to avoid the risk of viral infection and sensitization. The utilization rate of collagen extracted from edible fishes is lower than 65%, and the collagen cannot be completely absorbed and utilized by human bodies. It is therefore commonly used in food applications, a small fraction in cosmetics, but not in medical devices or more delicate tissue engineering products. Therefore, the current collagen can only be used in cosmetics and health care products, and cannot play the original biological function of the collagen at all.
Structurally, the structure of natural collagen in human body is very complicated, so that it is very difficult to express and prepare human collagen in large quantities by conventional means. The most common structural feature of collagen is the triple-helical structure formed by 3 peptide chains, i.e. the protein is formed by 3 a peptide chains in a right-handed supercoiled manner, and such triple-helical regions are called collagen regions. Each A peptide chain is composed of repeated Gly-X-Y (X, Y represents any amino acid residue except Gly, X is Pro, Y is Hyp) peptide segments on the molecular structure to form a left-hand helix, and 3 chains form a stable triple helix structure by taking the same axis as the center and in a right-hand supercoiling mode under the interaction of the amino acid residues. In organisms, collagen synthesis and modification starts from procollagen, undergoes chemical changes such as hydroxylation, glycosylation and mutual cross-linking, and is complexly regulated by various biological enzymes. Procollagen contains globular heads and tails in addition to collagen chains. Without these heads and tails, the collagen strands do not fold into the correct triple helix, thereby lacking the biological activity of collagen. Therefore, collagen prepared according to the original gene sequence cannot form the correct spatial structure in spontaneous tissues in vitro. Such difficulties have severely hampered the development and production of human collagen.
The conventional method for producing collagen is to treat animal-derived tissues by acid, alkali, and enzymatic hydrolysis to extract collagen derivatives. The collagen extracted by the methods loses the original biological activity and cannot be applied to the biomedical field to play a real function. Most of prepared collagen products have weak tensile strength; the pure collagen is degraded in vivo quickly, potential antigenicity may exist, meanwhile, due to the difference of the collagen source, the processing technology and the raw material proportion, the nutritional ingredients and the feeding value of the product are different, and the leather not only needs to contact with a plurality of chemical substances in the processing process, but also is easy to be infected by bacteria, thus the application of the collagen is severely limited. Although foreign research institutions have obtained human collagen-containing milk by breeding mice containing human collagen genes, the production cost is too high, the production period is too long, and large-scale production cannot be achieved.
Disclosure of Invention
The present invention is based in part on the following findings:
1. compared with the existing human collagen, the polypeptides C2R1T, C2R4T, C2R5T and C2R10T of the invention have equivalent or higher cell adhesion effect; and
2. the protein C2R1T for treating human papilloma virus infection can effectively inhibit HPV16, HPV18 and HPV58 high-risk human papilloma virus infection on an HPV pseudovirus infection model through experimental research. The experimental data show that the action mechanism of the antiviral protein is as follows: the C2R1T protein specifically binds to HPV L1 protein, thereby inhibiting viral infection of cells. The protein is found to act in the early stage of virus infection, namely the virus invasion stage through mechanism research, and has no inactivation effect.
3. Experiments prove that the C2R1T protein mainly depends on a core sequence R1P4GEPGLQGPAGPPGEKGEPGDDGPSGAEGPP (SEQ ID NO:18) to play a role in binding virus protein, and compared with other polypeptides from C2R1T, R1P4 has antiviral activity, and other sequences have NO good antiviral activity. The invention aims to provide an active protein capable of inhibiting human papilloma virus infection, and the virus is inhibited to change the disease process. The main idea is to block the continuous infection of the virus by inhibiting the invasion of HPV into target cells, finally reduce the virus amount and prevent/treat diseases such as cervical cancer and the like caused by the virus.
In view of the above-mentioned drawbacks of the prior art, the present invention provides:
1. a polypeptide comprising an N-terminal region and a C-terminal region,
wherein the N-terminal region comprises the N-terminal sequence of at least 48 consecutive amino acid residues of SEQ ID No. 13;
wherein the C-terminal region comprises the sequence GPPGPCCGGG shown as SEQ ID No. 14;
wherein preferably the polypeptide is a collagen polypeptide, preferably a human collagen polypeptide;
wherein preferably the N-terminal region comprises N repeats of an N-terminal sequence, N being an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
wherein preferably, when N is an integer of 2 or more, each N-terminal sequence is consecutive or spaced by 1 or more amino acid residues;
wherein preferably the N-terminal region is contiguous with or separated by 1 or more amino acid residues from the C-terminal region, provided that the polypeptide has cell adhesion activity; or
The polypeptide comprises (1) an amino acid sequence shown as SEQ ID NO. 18; (2) 18 by substitution, addition, deletion or deletion of 1 or more, for example, 2, 3, 4 or 5 amino acid residues; or (3) a sequence having 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% sequence identity to the amino acid sequence set forth in SEQ ID NO:18, provided that the polypeptide retains anti-HPV activity, preferably anti-HPV 16, 18 and/or 58 type activity.
2. The polypeptide of item 1, wherein the N-terminal sequence comprises a sequence selected from the group consisting of seq id no: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 or a sequence in any of these wherein 1 or more, for example 2, 3, 4 or 5 amino acid residues have been substituted, added, deleted or deleted.
3. The polypeptide according to item 1 or 2, comprising (1) the amino acid sequence of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, or SEQ ID No. 11; (2) an amino acid sequence in which 1 or more, for example 2, 3, 4 or 5, amino acid residues are substituted, added, deleted or deleted in the amino acid sequence of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or SEQ ID No. 11; or (3) a sequence having 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% sequence identity to the amino acid sequence of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or SEQ ID No.11, with the proviso that said polypeptide has cell adhesion activity or that said polypeptide retains anti-HPV activity, preferably anti-HPV 16, 18 and/or 58 type activity.
4. A polynucleotide encoding the polypeptide according to any one of items 1-3, wherein the polynucleotide is preferably SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, or SEQ ID No. 12.
5. An expression vector comprising the polynucleotide according to item 4.
6. A host cell comprising the expression vector according to item 5, wherein the host cell is preferably E.coli.
7. A method for producing a polypeptide according to any one of claims 1-3, comprising:
(1) culturing the host cell according to item 6 in a production medium and producing the polypeptide;
(2) harvesting and purifying the polypeptide; and
(3) optionally cleaving the polypeptide with an enzyme.
8. A composition comprising a polypeptide according to any of claims 1-3, wherein the composition is preferably a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic or a nutraceutical, preferably the pharmaceutical composition is an external preparation, preferably an external spread preparation, such as an external gel or an external infiltrate preparation; wherein preferably the topical gel further comprises a pharmaceutically acceptable carrier vehicle, and the topical infiltration formulation further comprises a sterile medical cotton ball having the polypeptide of any of items 1-3.
9. Use of the polypeptide of any one of items 1 to 3, the polynucleotide of item 4, the expression vector of item 5, the host cell of item 6, or the composition of item 8 in the manufacture of a composition, preferably a medical device, a tissue engineering product, a cosmetic product, a nutraceutical product, or use of the polypeptide of any one of items 1 to 3, the polynucleotide of item 4, the expression vector of item 5, the host cell of item 6, or the composition of item 8 in the manufacture of a product for preventing or treating an HPV infection or a disease caused by an HPV infection, wherein preferably the HPV infection is positive for detection, and the disease caused by HPV persistent infection is a disease caused by HPV persistent infection, preferably cervical neoplasia, condyloma acuminatum, or cervical cancer, wherein HPV is preferably resistant to HPV16, 18, and/or HPV type 58.
10. Use of the polypeptide of any one of items 1-3, the polynucleotide of item 4, the expression vector of item 5, the host cell of item 6, or the composition of item 8 to promote cell adhesion or inhibit HPV or bind HPV L1 protein in vitro, wherein HPV is preferably anti-HPV type 16, 18, and/or 58.
11. A method for preventing or treating HPV infection or a disease caused by HPV infection, comprising the steps of: covering or applying the polypeptide according to any one of items 1-3 or the composition according to item 8 to the cervical region of a patient, wherein preferably the HPV infection means positive HPV detection and the disease caused by HPV infection means a disease caused by HPV persistent infection, preferably cervical neoplasia, condyloma acuminatum or cervical cancer, wherein HPV is preferably anti-HPV 16, 18 and/or 58.
12. The polypeptide of any one of items 1 to 3, the polynucleotide of item 4, the expression vector of item 5, the host cell of item 6, or the composition of item 8, for use in promoting cell adhesion or in inhibiting HPV or preventing or treating HPV infection or a disease caused by HPV infection or binding to HPV L1 protein, wherein preferably the HPV infection is positive for HPV detection and the disease caused by HPV infection is a disease caused by HPV persistent infection, preferably cervical neoplasia, condyloma acuminatum or cervical cancer, wherein HPV is preferably anti-HPV 16, 18 and/or 58.
Compared with the prior art, the invention has the following characteristics:
(1) the selected collagen sequence is a type II collagen functional region sequence obtained through long-term screening and optimization for the first time;
(2) the Escherichia coli expression system is adopted, the large-scale amplification is suitable, one round of fermentation can be completed within 20 hours, the production cost is very low, and the yield is very high due to the codon optimization of Escherichia coli on a gene sequence and the selection of a2 XYT culture medium;
(3) the produced humanized II type collagen has good hydrophilicity and stability, the amino acid composition of the humanized II type collagen is 100 percent same as the corresponding part of the amino acid sequence of the natural collagen, and the humanized II type collagen can not generate immunological rejection and anaphylactic reaction when being applied to a human body and can be widely applied to the industries of biological medicine and cosmetics;
(4) the product of the invention has biological activity reaching or even exceeding the biological activity of natural protein of human body through activity detection, is the optimized collagen design scheme reported at present, can perform the function of the natural protein in human body, and achieves the purpose of real product application.
(5) The protein C2R1T for treating human papilloma virus infection can effectively inhibit HPV16, HPV18 and HPV58 high-risk human papilloma virus infection on an HPV pseudovirus infection model through experimental research.
Drawings
FIG. 1 is a plasmid map constructed by the vectors of the invention, pET32a-C2R1T, pET32a-C2R4T, PET32a-C2R5T and PET32a-C2R 10T.
FIG. 2 is an electrophoretogram of a target protein obtained after purification and enzyme digestion of C2R1T protein of the invention; electrophoresis of the C2R1T protein detected a molecular weight of about 30kDa, corresponding to a protein comprising the amino acid sequence of SEQ ID No. 5.
FIG. 3 is an electrophoretogram of a target protein obtained after purification and digestion of C2R4T protein of the present invention; electrophoresis of the C2R4T protein detected a molecular weight of approximately 36kDa, corresponding to a protein comprising the amino acid sequence of SEQ ID No. 7.
FIG. 4 is an electrophoretogram of a target protein obtained after purification and digestion of C2R5T protein of the present invention; electrophoresis of the C2RT1 protein detected a molecular weight of approximately 34kDa, corresponding to a protein comprising the amino acid sequence of SEQ ID NO. 9.
FIG. 5 is an electrophoretogram of a target protein obtained after purification and digestion of C2R10T protein of the present invention; the electrophoretic detection of the C2RT1 protein has a molecular weight of approximately 25kDa, corresponding to a protein comprising the amino acid sequence of SEQ ID NO. 11.
FIG. 6 shows the results of the bioactivity test of the C2R1T protein of the present invention compared with human collagen.
FIG. 7 shows the results of the bioactivity test of the C2R4T protein of the present invention compared with human collagen.
FIG. 8 shows the results of the bioactivity assay of C2R5T protein of the present invention compared with human collagen.
FIG. 9 shows the results of the bioactivity assay of C2R10T protein of the present invention compared with human collagen.
FIGS. 10A-C: C2R1T all had inhibitory activity against HPV16, 18 and 58, with triplicate wells per sample and data expressed as mean ± Standard Deviation (SD).
FIG. 11: C2R1T repetitive unit sequence and related polypeptide R1P1, R1P2, R1P3 and R1P4 sequence.
FIGS. 12A-D: the virus inhibition experiment shows that R1P4 has the inhibition activity, the other three polypeptides have no inhibition activity, and IC50 cannot be calculated.
FIG. 13: the results of the TIME-REMOVAL experiment showed that C2R1T did not act on the target cells. The antiviral effect of the exclusins is due to targeting of the target cells.
FIG. 14: inactivation experiments show that the C2R1T protein has no inactivation activity.
FIG. 15: the ELISA results show that C2R1T and R1P4 can bind to HPV16 type L1, JB01 is used as a positive control, and BSA is used as a negative control.
Detailed Description
Further description is provided below to facilitate understanding of the invention.
As used herein, "medical instrument" refers to instruments, devices, instruments, in vitro diagnostic reagents and calibrators, materials, and other similar or related items used, directly or indirectly, in the human body.
As used herein, "tissue engineering product" refers to a product used for tissue engineering. Tissue engineering is an emerging discipline for the construction of tissues or organs in vitro or in vivo, combining cell biology and material science.
In the present invention, type II collagen sequences are selected as the sequences to be optimized for screening. The sequence of human collagen type II is the NCBI reference sequence: NM-001844.4 (SEQ ID No.13), see
Figure BDA0001968349440000081
The amino acid sequences selected in the present invention are underlined in bold in the above sequences. The applicant has found, through extensive studies, that the above sequence selected achieves a better adhesion effect than the commercial human collagen or other sequences in SEQ ID No. 13. In the present invention, the polypeptide is not the full-length sequence of SEQ ID No. 13.
The present invention is based in part on the following findings: a polypeptide comprising the N-terminal region of at least 48 consecutive amino acid residues in SEQ ID No.13 and the C-terminal region of the sequence GPPGPCCGGG shown in SEQ ID No.14 is able to achieve a better adhesion effect than commercial human collagen, as demonstrated in the examples. The continuous amino acid residues constituting the N-terminal region can be appropriately selected by those skilled in the art. For example, contiguous amino acid residues can be 48-100, 50-72, 54-57, 48-72, and so forth in length.
In the present invention, several specific sequences of N-terminal regions were tested:
(1)GPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQ(SEQ IDNo.1)；
(2)GDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKA(SEQ ID No.2)；
(3)GPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPT GVTGPKGARGAQGPP(SEQ IDNo.3)；
(4)GEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGL KGHRGFT(SEQ ID No.4)。
in the present invention, the C-terminal amino acid sequence may be GPPGPCCGGG (SEQ ID No.14), which is a terminal peptide segment that enhances collagen activity.
The polypeptide can be humanized type II collagen C2R1T in the present specification, and has a single chain structure comprising 226 amino acids, a basic repeating unit of GPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEG PPGPQ (SEQ ID No.1), a human collagen type II peptide segment, and a C-terminal amino acid sequence of GPPGPCCGGG (SEQ ID No.14), which is a terminal sequence peptide segment for enhancing collagen activity. The amino acid sequence of C2R1T is as follows: GPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGPPGPCCGGG (SEQ ID No. 5). The DNA sequence of C2R1T is as follows: GGTCCTAGTGGTAAAGATGGTCCGAAAGGTGCACGTGGTGATAGTGGTCCGCCGGGTCGTGCAGGTGAACCGGGTCTGCAGGGTCCGGCAGGTCCGCCTGGTGAGAAAGGTGAACCGGGCGATGATGGGCCGAGCGGTGCCGAAGGTCCTCCTGGTCCGCAAGGTCCGTCTGGTAAAGATGGGCCGAAAGGTGCCAGAGGGGATAGTGGGCCGCCGGGTAGAGCAGGTGAACCTGGGCTGCAGGGTCCTGCAGGTCCGCCAGGTGAAAAAGGGGAACCAGGTGATGATGGACCAAGCGGTGCAGAAGGTCCGCCGGGCCCTCAGGGTCCTAGCGGTAAAGATGGCCCGAAAGGTGCGAGAGGTGATAGCGGACCGCCGGGAAGAGCAGGAGAACCAGGACTGCAGGGACCGGCAGGTCCTCCGGGTGAAAAAGGTGAACCAGGTGACGATGGGCCGAGTGGTGCAGAAGGACCGCCGGGTCCGCAGGGACCAAGCGGCAAAGACGGACCGAAAGGAGCAAGAGGAGATAGTGGACCGCCGGGCCGGGCAGGTGAACCAGGCTTACAGGGTCCGGCGGGTCCGCCAGGAGAAAAAGGGGAGCCGGGTGATGATGGGCCAAGCGGAGCAGAAGGACCTCCGGGTCCGCAAGGACCTCCAGGTCCATGTTGTGGAGGTGGG (SEQ ID No. 6).
The polypeptide can be human collagen C2R4T, and has single chain structure comprising 298 amino acids, GDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKA (SEQ ID No.2) as basic repeating unit, GPPGPCCGGG (SEQ ID No.14) as C-terminal amino acid sequence and peptide segment as collagen activity enhancing end sequence. The amino acid sequence of C2R4T is as follows: GDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKAGDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKAGDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKAGDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKAGPPGPCCGGG (SEQ ID No. 7). The DNA sequence of C2R4T is as follows: GGTGATCCGGGTCGTCCGGGTGAACCGGGTCTGCCTGGTGCGCGTGGTCTGACAGGTCGTCCGGGAGATGCGGGGCCGCAGGGTAAAGTTGGGCCGAGCGGGGCGCCGGGTGAAGATGGTCGTCCGGGCCCTCCGGGTCCGCAAGGTGCAAGAGGTCAGCCTGGTGTTATGGGTTTTCCTGGTCCGAAAGGTGCAAATGGGGAGCCGGGTAAAGCAGGTGATCCGGGAAGACCGGGTGAACCTGGTCTGCCTGGCGCCAGAGGGTTAACAGGTCGTCCTGGTGATGCAGGTCCGCAGGGTAAGGTTGGTCCGTCGGGAGCACCGGGTGAAGACGGTAGACCGGGTCCGCCTGGGCCGCAAGGTGCTAGAGGTCAGCCGGGTGTGATGGGTTTTCCGGGTCCGAAAGGGGCAAATGGGGAACCGGGTAAAGCGGGGGACCCTGGGCGTCCAGGTGAACCTGGCCTGCCGGGTGCAAGAGGATTAACAGGTCGGCCGGGGGATGCAGGTCCTCAAGGGAAAGTGGGTCCGAGCGGTGCACCGGGTGAGGATGGGAGACCGGGTCCTCCGGGGCCACAAGGTGCACGTGGTCAGCCGGGGGTGATGGGTTTCCCGGGGCCTAAAGGGGCAAACGGTGAGCCGGGTAAGGCAGGTGATCCAGGTCGGCCGGGTGAACCAGGTCTGCCGGGTGCTCGTGGTTTAACAGGTCGCCCGGGTGATGCGGGTCCTCAGGGTAAAGTGGGTCCTAGCGGGGCACCTGGAGAAGATGGGCGTCCGGGTCCTCCTGGGCCGCAGGGCGCACGTGGTCAACCTGGTGTTATGGGGTTTCCTGGTCCTAAAGGTGCAAACGGGGAACCGGGCAAAGCAGGACCGCCGGGTCCGTGTTGTGGTGGTGGT (SEQ ID No. 8).
The polypeptide can be human collagen C2R5T, and has single chain structure comprising 238 amino acids, GPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPK GARGAQGPP (SEQ ID No.3) as basic repeating unit, GPPGPCCGGG as C terminal amino acid sequence (SEQ ID No.14), and peptide segment as collagen activity enhancing end sequence. The amino acid sequence of C2R5T is as follows: GPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPKGARGAQGPPGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPKGARGAQGPPGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPKGARGAQGPPGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPKGARGAQGPPGPPGPCCGGG (SEQ ID No. 9). The DNA sequence of C2R5T is as follows:
GGTCCTCCTGGTGCAGATGGTCAGCCGGGTGCAAAAGGTGAACAGGGTGAAGCAGGTCAGAAAGGTGATGCAGGGGCACCGGGTCCGCAGGGTCCTAGTGGTGCACCGGGTCCTCAGGGTCCGACCGGTGTAACCGGTCCGAAAGGAGCAAGAGGTGCACAGGGACCGCCGGGTCCACCGGGTGCAGATGGACAGCCTGGTGCAAAAGGGGAACAGGGTGAGGCAGGTCAGAAGGGTGATGCAGGTGCACCAGGACCGCAGGGACCGAGCGGTGCACCAGGTCCTCAGGGCCCAACCGGGGTTACCGGTCCGAAGGGGGCAAGAGGAGCACAGGGACCACCGGGACCACCGGGTGCTGATGGTCAGCCTGGAGCAAAAGGAGAACAGGGGGAAGCAGGGCAAAAAGGAGATGCAGGTGCGCCGGGACCGCAGGGTCCAAGTGGAGCACCAGGACCACAAGGACCGACCGGTGTGACGGGTCCGAAAGGGGCAAGAGGGGCACAGGGACCTCCAGGTCCGCCGGGTGCAGACGGTCAGCCTGGTGCTAAAGGGGAACAAGGAGAAGCAGGACAAAAAGGAGACGCAGGTGCGCCAGGACCGCAAGGTCCGAGCGGTGCTCCAGGTCCACAGGGTCCCACCGGTGTTACAGGTCCAAAAGGGGCACGCGGAGCACAGGGGCCGCCAGGTCCTCCTGGACCTTGTTGTGGTGGTGGT(SEQ ID No.10)。
the polypeptide can be human collagen C2R10T, and has single chain structure comprising 202 amino acids, GEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFT basic repeating unit (SEQ ID No.4), human collagen II peptide segment, and GPPGPCCGGG C terminal amino acid sequence. The amino acid sequence of C2R10T is as follows:
GEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGPPGPCCGGG (SEQ ID No. 11). The DNA sequence of C2R10T is as follows:
GGTGAAGCAGGTGCGCAGGGTCCGATGGGGCCGAGTGGTCCTGCGGGTGCAAGAGGAATTCAGGGTCCGCAGGGGCCGAGAGGGGATAAAGGAGAAGCAGGGGAACCGGGTGAAAGAGGGCTGAAAGGTCATAGAGGGTTTACGGGTGAAGCAGGAGCACAGGGACCGATGGGACCGAGCGGACCGGCAGGAGCAAGAGGAATACAGGGACCGCAGGGACCGAGAGGAGATAAAGGTGAAGCAGGGGAGCCGGGAGAAAGAGGACTGAAAGGACATAGAGGATTTACAGGAGAAGCAGGAGCGCAGGGACCGATGGGGCCAAGCGGACCTGCAGGAGCACGGGGAATACAGGGTCCGCAAGGACCGAGAGGGGACAAAGGAGAAGCGGGAGAACCGGGAGAGAGAGGACTGAAGGGACATAGAGGGTTCACGGGTGAAGCCGGAGCACAAGGACCGATGGGTCCGAGCGGGCCGGCAGGTGCAAGAGGTATACAGGGACCACAGGGACCGCGGGGAGATAAAGGAGAGGCAGGAGAACCGGGTGAGAGAGGATTAAAAGGACATCGGGGATTTACCGGTCCGCCGGGACCATGTTGTGGTGGGGGG(SEQ ID No.12)。
the present invention is also based in part on the following findings: the protein C2R1T for treating human papilloma virus infection can effectively inhibit the infection of HPV16, HPV18 and HPV58 high-risk human papilloma virus on an HPV pseudovirus infection model through experimental research. The experimental data show that the action mechanism of the antiviral protein is as follows: the C2R1T protein specifically binds to HPV L1 protein, thereby inhibiting viral infection of cells. The protein is found to act in the early stage of virus infection, namely the virus invasion stage through mechanism research, and has no inactivation effect. Experiments prove that the C2R1T protein mainly depends on a core sequence R1P4GEPGLQGPAGPPGEKGEPGDDGPSGAEGPP (SEQ ID NO:18) to play a role in binding virus protein, and compared with other polypeptides from C2R1T, R1P4 has antiviral activity, and other sequences have NO good antiviral activity. The invention aims to provide an active protein capable of inhibiting human papilloma virus infection, and the virus is inhibited to change the disease process. The main idea is to block the continuous infection of the virus by inhibiting the invasion of HPV into target cells, finally reduce the virus amount and prevent/treat diseases such as cervical cancer and the like caused by the virus.
Herein, the polypeptide may include an amino acid sequence in which 1 or more, preferably 2, 3, 4 or 5 amino acid residues are substituted, added, deleted or inserted in the amino acid sequence shown in any one of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No. 18 or an amino acid sequence having 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% sequence identity with the amino acid sequence shown in any one of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No. 18.
Amino acid addition refers to the addition of an amino acid at the C-terminus or N-terminus of an amino acid sequence, e.g., any of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No. 18, as long as the polypeptide has cell adhesion activity or the polypeptide retains anti-HPV activity, preferably anti-HPV 16 activity.
Amino acid substitution refers to the substitution of an amino acid residue at a position in an amino acid sequence, such as the sequence of any one of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No. 18, with another amino acid residue, as long as the polypeptide has cell adhesion activity or the polypeptide retains anti-HPV activity, preferably anti-HPV 16 activity.
Amino acid insertion refers to the insertion of amino acid residues at appropriate positions in an amino acid sequence, such as any of the sequences of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No. 18, which may also be adjacent to each other in whole or in part, or none of the inserted amino acids, as long as the polypeptide has cell adhesion activity or the polypeptide retains anti-HPV activity, preferably anti-HPV 16 activity.
Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from an amino acid sequence, such as the sequence of any one of SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No. 18, as long as the polypeptide has cell adhesion activity or the polypeptide retains anti-HPV activity, preferably anti-HPV 16 activity.
In the present invention, the substitution may be a conservative amino acid substitution, which refers to a substitution with SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, and SEQ ID NO:18, or a peptide wherein 3, preferably 2 or 1 amino acid residues are substituted with amino acids having similar or similar properties. These conservative variant peptides can be generated by amino acid substitutions according to table 1.
Table 1: conservative substitution of amino acid
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
All amino acids in the polypeptide sequences herein can be L-form amino acids, one or more (e.g., 2-5, 2-4, or 2-3) of which can also be substituted with D-form amino acids in conformation, artificially modified amino acids, naturally occurring unusual amino acids, and the like, to enhance the bioavailability, stability, and/or antiviral activity of the polypeptide. Wherein the D-form amino acid is an amino acid corresponding to the L-form amino acid constituting the protein; the artificially modified amino acid refers to common L-type amino acid which is modified by methylation, phosphorylation and the like and forms protein; the rare amino acids existing in nature include unusual amino acids constituting proteins and amino acids not constituting proteins, such as 5-hydroxylysine, methylhistidine, gamma-aminobutyric acid, homoserine and the like.
In the present invention, the humanization of the type II collagen can be performed by a method which is conventional in the art. For example, it can be produced by the following steps: (1) constructing escherichia coli genetic engineering bacteria; (2) fermenting and culturing the escherichia coli genetic engineering bacteria; (3) induction and expression of humanized type II collagen; and (4) purification and optional enzymatic cleavage of the humanized type II collagen.
In the step (1), the construction of the engineered Escherichia coli can be carried out as follows: (1) carrying out codon optimization and splicing recombination on a DNA fragment of a gene spiral region of the human type II collagen by using a PCR method to finally obtain a target gene fragment; (2) inserting the obtained target gene fragment into a PET-32a expression vector to obtain a recombinant expression plasmid; (3) transferring the recombinant expression plasmid into an escherichia coli competent cell BL21(DE3), and screening to obtain the positive escherichia coli genetic engineering bacteria.
In steps (2) and (3), the fermentation culture of the genetically engineered Escherichia coli and the induction and expression of the humanized type II collagen can be performed as follows: (1) picking out the optimized single colony of the escherichia coli genetic engineering bacteria from the LAB plate, and placing the single colony in 10ml of LB culture medium at 37 ℃ and 220rpm for culturing for 12-16 hours; (2) and (3) mixing the bacterial liquid according to the proportion of 1:100 inoculating into 2 XYT medium, culturing at 37 deg.C for about 3 hr until OD reaches600At 0.4-0.6, adding IPTG with final concentration of 0.5mM for induction, culturing at 16 deg.C for 20 hr, and centrifuging to collect thallus.
In step (4), the purification and cleavage of the humanized type II collagen polypeptide may be performed as follows: (1) with phosphate buffer (40mM NaH)2PO3500mM NaCl, pH7.8), resuspending the bacteria, sonicating, centrifuging, and collecting the supernatant; (2) combining NI-NTA affinity column with humanized II type collagen, rinsing hybrid protein with 10mM imidazole, adding Prescission protease (PPase) protease at 4 deg.C, and performing enzyme digestion on 16h column to obtain the target collagen polypeptide.
The host cell may be a eukaryotic cell, such as a fungus and a yeast, a prokaryotic cell, such as a bacterium of the family Enterobacteriaceae. It is understood that one skilled in the art may substitute the above E.coli strain for other expression strains as host cells.
Examples
The following examples are provided to illustrate the invention. It is to be understood by persons skilled in the art that the embodiments are merely illustrative and not restrictive. The invention is limited only by the scope of the appended claims.
Example 1: construction and expression of humanized type II collagen polypeptide
Construction and expression of C2R1T gene expression vector
1. The full-length gene sequence of human collagen C2R1T used in example 1 is shown as SEQ ID No. 6. The sequence has been codon optimized for the codons of E.coli.
The C2R1T gene has a full length of 678bp, the optimized C2R1T codon gene sequence SEQ ID No.6 is assigned to Shanghai Huajin Biotechnology Co., Ltd for gene fragment synthesis, and the synthesized C2R1T gene fragment is inserted into the PET32a expression vector through the enzyme cutting sites of BamHI (NEB Co., Ltd.: R0136L) and Xho I (NEB Co., Ltd.: R0146L). Coli competent cells BL21(DE3) (Merck) were transformed with the successfully constructed expression plasmid. The specific process is as follows: 1: mu.l of this plasmid was taken in 100. mu.l of E.coli competent cells BL21(DE3) and left to stand on ice for 30 min. 2: the mixture was heat-shocked in a 42 ℃ water bath for 90s and then quickly placed on ice for 2 min. 3: to the mixture was added 600. mu.l of non-resistant LB, and the mixture was incubated at 37 ℃ for 1 hour at 220 rpm. 4: 200. mu.l of the suspension was applied evenly to LB plates containing ampicillin resistance (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100. mu.g/ml ampicillin). 5: the plates were cultured upside down in an incubator at 37 ℃ for about 20 hours until clearly visible colonies grew.
3. Monoclonal colonies were picked from the transformed LB plates and cultured in 10ml LB medium (containing 100. mu.g/ml ampicillin) for 12-16 h, then transferred to 2 XYT medium (16G/L peptone, 10G/L yeast extract, 5G/L sodium chloride) at a ratio of 1:100 for amplification culture, cultured at 37 ℃ and 220rpm until OD600 of the bacterial solution was 0.4-0.6, and then induced to express by adding 0.5mM IPTG (Sigma; cat # I5502-1G) at a final concentration, and cultured at 18 ℃ and 180rpm for 20 h. Finally, the thalli are collected by centrifugation and stored at the temperature of minus 20 ℃ or immediately purified in the next step.
4. About 50ml of the suspension was resuspended (1L) in phosphate buffer (pH 7.8) (40mM sodium dihydrogen phosphate, 500mM sodium chloride) and the pellet was disrupted by a high-pressure disruption apparatus (New Ganoderma organism), and then centrifuged at 13000rpm for 30min to separate the soluble protein from the inclusion bodies sufficiently.
5. Binding buffer (40mM NaH) was applied in a 5 column volume2PO3500mM NaCl, pH7.8) balance Ni-NTA (Qiagen, Cat #: 30210) An affinity column. Then adding protein supernatant and incubating for 0.5-1h at 4 ℃ to ensure that the target protein is fully combined on the column material. 200ml of washing buffer (10mM imidazole, 40mM NaH) containing 10mM imidazole (Sigma) was added2PO3500mM NaCl, pH7.8) rinsing the hetero-proteins. Finally, adding a proper amount of Prescission Protease (PPase for short) (Sigma, SAE0045) Protease with His label, incubating for 16h at 4 ℃, and collecting the transudate, namely the target collagen from which the carrier protein is removed. The product was dialyzed overnight and lyophilized to a dry powder for use.
6. The purity of the obtained C2R1T protein is detected by SDS-PAGE, which comprises adding 10 μ l of 5 Xprotein loading buffer (250mM Tris-HCl (pH:6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β -mercaptoethanol) into 40 μ l of purified protein solution, boiling in 100 deg.C boiling water for 10min, adding 10 μ l of SDS-PAGE protein gel into each well, running at 80V for 2h, staining protein with Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20min, decolorizing with protein decolorizing solution (10% acetic acid, 5% ethanol), and comparing with human native collagen to measure protein activity.
Construction and expression of C2R4T gene expression vector
1. The full-length gene sequence of the human collagen C2R1T is shown as SEQ ID No. 8. The sequence has been codon optimized for the codons of E.coli.
The C2R4T gene has a total length of 894bp, and the optimized C2R4T codon gene sequence is used to make synthesis of gene fragment by Shanghai Huajin Biotechnology Co., Ltd, and the synthesized C2R1T gene fragment is inserted into the PET32a expression vector through enzyme cutting sites of BamH I (NEB Co., Ltd.: R0136L) and Xho I (NEB Co., Ltd.: R0146L). Coli competent cells BL21(DE3) (Merck) were transformed with the successfully constructed expression plasmid. The specific process is as follows: 1: mu.l of this plasmid was taken in 100. mu.l of E.coli competent cells BL21(DE3) and left to stand on ice for 30 min. 2: the mixture was heat-shocked in a 42 ℃ water bath for 90s and then quickly placed on ice for 2 min. 3: to the mixture was added 600. mu.l of non-resistant LB, and the mixture was incubated at 37 ℃ for 1 hour at 220 rpm. 4: 200. mu.l of this strain was applied evenly to LAB plates containing ampicillin resistance (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100. mu.g/ml ampicillin). 5: the plates were cultured upside down in an incubator at 37 ℃ for about 20 hours until clearly visible colonies grew.
3. Monoclonal colonies were picked from the transformed LB plates and cultured in 10ml LB medium (containing 100. mu.g/ml ampicillin) for 12-16 h, then transferred to 2 XYT medium (16G/L peptone, 10G/L yeast extract, 5G/L sodium chloride) at a ratio of 1:100 for amplification culture, cultured at 37 ℃ and 220rpm until OD600 of the bacterial solution was 0.4-0.6, and then induced to express by adding 0.5mM IPTG (Sigma; cat # I5502-1G) at a final concentration, and cultured at 18 ℃ and 180rpm for 20 h. Finally, the thalli are collected by centrifugation and stored at the temperature of minus 20 ℃ or immediately purified in the next step.
4. About 50ml of the suspension was resuspended (1L) in phosphate buffer (pH 7.8) (40mM sodium dihydrogen phosphate, 500mM sodium chloride) and the pellet was disrupted by a high-pressure disruption apparatus (New Ganoderma organism), and then centrifuged at 13000rpm for 30min to separate the soluble protein from the inclusion bodies sufficiently.
5. Binding buffer (40mM NaH) was applied in a 5 column volume2PO3500mM NaCl, pH7.8) equilibrium Ni-NTA affinity column (Qiagen, cat #: 30210). Then adding protein supernatant and incubating for 0.5-1h at 4 ℃ to ensure that the target protein is fully combined on the column material. 200ml of washing buffer (10mM imidazole, 40mM NaH) containing 10mM imidazole (Sigma) was added2PO3500mM NaCl, pH7.8) rinsing the hetero-proteins. Finally adding proper amount of the mixtureAnd incubating the protein with His-tagged Prescission Protease (PPase for short) (Sigma, SAE0045) at 4 ℃ for 16h, and collecting the transudate, namely the target collagen from which the carrier protein is removed. The product was dialyzed overnight and lyophilized to a dry powder for use.
6. The purity of the obtained C2R4T protein is detected by SDS-PAGE, which comprises adding 10 μ l of 5 Xprotein loading buffer (250mM Tris-HCl (pH:6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β -mercaptoethanol) into 40 μ l of purified protein solution, boiling in 100 deg.C boiling water for 10min, adding 10 μ l of SDS-PAGE protein gel into each well, running at 80V for 2h, staining protein with Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20min, decolorizing with protein decolorizing solution (10% acetic acid, 5% ethanol), and comparing with human native collagen to measure protein activity.
Construction and expression of C2R5T gene expression vector
1. The full-length gene sequence of the human collagen C2R1T is shown as SEQ ID No. 10. The sequence has been codon optimized for the codons of E.coli.
The C2R5T gene has a total length of 715bp, and the optimized C2R5T codon gene sequence is used to consign the synthesis of gene fragments by Shanghai Huajin Biotechnology Co., Ltd, and the synthesized C2R1T gene fragment is inserted into the PET32a expression vector through enzyme cutting sites of BamH I (NEB Co., Ltd.: R0136L) and Xho I (NEB Co., Ltd.: R0146L). Coli competent cells BL21(DE3) (Merck) were transformed with the successfully constructed expression plasmid. The specific process is as follows: 1: mu.l of this plasmid was taken in 100. mu.l of E.coli competent cells BL21(DE3) and left to stand on ice for 30 min. 2: the mixture was heat-shocked in a 42 ℃ water bath for 90s and then quickly placed on ice for 2 min. 3: to the mixture was added 600. mu.l of non-resistant LB, and the mixture was incubated at 37 ℃ for 1 hour at 220 rpm. 4: 200. mu.l of this strain was applied evenly to LAB plates containing ampicillin resistance (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100. mu.g/ml ampicillin). 5: the plates were cultured upside down in an incubator at 37 ℃ for about 20 hours until clearly visible colonies grew.
3. Monoclonal colonies were picked from the transformed LB plates and cultured in 10ml LB medium (containing 100. mu.g/ml ampicillin) for 12-16 h, then transferred to 2 XYT medium (16G/L peptone, 10G/L yeast extract, 5G/L sodium chloride) according to the ratio of 1:100 for amplification culture, cultured at 37 ℃ and 220rpm until the bacterial liquid OD600 was 0.4-0.6, and then added with 0.5mMIPTG (Sigma; cat # I5502-1G) to induce expression under the induction conditions of 18 ℃ and 180rpm for 20 h. Finally, the thalli are collected by centrifugation and stored at the temperature of minus 20 ℃ or immediately purified in the next step.
4. About 50ml of the suspension was resuspended (1L) in phosphate buffer (pH 7.8) (40mM sodium dihydrogen phosphate, 500mM sodium chloride) and the pellet was disrupted by a high-pressure disruption apparatus (New Ganoderma organism), and then centrifuged at 13000rpm for 30min to separate the soluble protein from the inclusion bodies sufficiently.
5. Binding buffer (40mM NaH) was applied in a 5 column volume2PO3500mM NaCl, pH7.8) balance Ni-NTA (Qiagen, Cat #: 30210) An affinity column. Then adding protein supernatant and incubating for 0.5-1h at 4 ℃ to ensure that the target protein is fully combined on the column material. 200ml of washing buffer (10mM imidazole, 40mM NaH) containing 10mM imidazole (Sigma) was added2PO3500mM NaCl, pH7.8) rinsing the hetero-proteins. Finally, adding a proper amount of Prescission Protease (PPase for short) Protease (Sigma, SAE0045) with His label, incubating for 16h at 4 ℃, and collecting the transudate, namely the target collagen from which the carrier protein is removed. The product was dialyzed overnight and lyophilized to a dry powder for use.
6. The purity of the obtained C2R5T protein is detected by SDS-PAGE, which comprises adding 10 μ l of 5 Xprotein loading buffer (250mM Tris-HCl (pH:6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β -mercaptoethanol) into 40 μ l of purified protein solution, boiling in 100 deg.C boiling water for 10min, adding 10 μ l of SDS-PAGE protein gel into each well, running at 80V for 2h, staining protein with Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20min, decolorizing with protein decolorizing solution (10% acetic acid, 5% ethanol), and comparing with human native collagen to measure protein activity.
Construction and expression of C2R10T gene expression vector
1. The full-length gene sequence of the human collagen C2R1T is shown as SEQ ID No. 12. The sequence has been codon optimized for the codons of E.coli.
The C2R10T gene has a full length of 606bp, and the optimized C2R10T codon gene sequence is used to order Shanghai Huajin Biotechnology Co., Ltd for gene fragment synthesis, and the synthesized C2R1T gene fragment is inserted into the PET32a expression vector through enzyme cutting sites of BamH I (NEB Co., Ltd.: R0136L) and Xho I (NEB Co., Ltd.: R0146L). Coli competent cells BL21(DE3) (Merck) were transformed with the successfully constructed expression plasmid. The specific process is as follows: 1: mu.l of this plasmid was taken in 100. mu.l of E.coli competent cells BL21(DE3) and left to stand on ice for 30 min. 2: the mixture was heat-shocked in a 42 ℃ water bath for 90s and then quickly placed on ice for 2 min. 3: to the mixture was added 600. mu.l of non-resistant LB, and the mixture was incubated at 37 ℃ for 1 hour at 220 rpm. 4: 200. mu.l of this strain was applied evenly to LAB plates containing ampicillin resistance (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100. mu.g/ml ampicillin). 5: the plates were cultured upside down in an incubator at 37 ℃ for about 20 hours until clearly visible colonies grew.
3. Monoclonal colonies were picked from the transformed LB plates and cultured in 10ml LB medium (containing 100. mu.g/ml ampicillin) for 12-16 h, then transferred to 2 XYT medium (16G/L peptone, 10G/L yeast extract, 5G/L sodium chloride) at a ratio of 1:100 for amplification culture, cultured at 37 ℃ and 220rpm until OD600 of the bacterial solution was 0.4-0.6, and then induced to express by adding 0.5mM IPTG (Sigma; cat # I5502-1G) at a final concentration, and cultured at 18 ℃ and 180rpm for 20 h. Finally, the thalli are collected by centrifugation and stored at the temperature of minus 20 ℃ or immediately purified in the next step.
4. About 50ml of the suspension was resuspended (1L) in phosphate buffer (pH 7.8) (40mM sodium dihydrogen phosphate, 500mM sodium chloride) and the pellet was disrupted by a high-pressure disruption apparatus (New Ganoderma organism), and then centrifuged at 13000rpm for 30min to separate the soluble protein from the inclusion bodies sufficiently.
5. Binding buffer (40mM NaH) was applied in a 5 column volume2PO3500mM NaCl, pH7.8) equilibrium Ni-NTA affinity column (Qiagen, cat #: 30210). Then adding protein supernatant and incubating for 0.5-1h at 4 ℃ to ensure that the target protein is fully combined on the column material. 200ml of washing buffer (10mM imidazole, 40mM NaH) containing 10mM imidazole (Sigma) was added2PO3500mM NaCl, pH7.8) rinsing the hetero-proteins. Finally, adding a proper amount of Prescission Protease (PPase for short) Protease (Sigma, SAE0045) with His label, incubating for 16h at 4 ℃, and collecting the transudate, namely the target collagen from which the carrier protein is removed. The product was dialyzed overnight and lyophilized to a dry powder for use.
6. The purity of the obtained C2R10T protein is detected by SDS-PAGE, which comprises the steps of adding 10 mul of 5x protein loading buffer (250mM Tris-HCl (pH:6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β -mercaptoethanol) into 40 mul of purified protein solution, boiling in 100 ℃ boiling water for 10min, adding 10 mul of protein gel into SDS-PAGE per well, running at 80V for 2h, staining proteins for 20min by Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid), decolorizing by protein decolorizing solution (10% acetic acid, 5% ethanol), and comparing with human native collagen to measure protein activity.
Results
FIGS. 2-5 show electrophoretograms that give C2R1T, C2R4T, C2R5T, and C2R10T apparent molecular weights of 30kDa, 36kDa, 34kDa, and 25kDa, respectively, and polypeptides having molecular weights corresponding to the amino acid sequences of SEQ ID NOS 5, 7, 9, and 11, respectively.
Example 2 Activity assays of C2R1T, C2R4T, C2R5T and C2R10T proteins
Methods for detecting Collagen activity may be found in Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-like proteins Based on the Cell additive and Crosslinking Sequences Derived from native Collagen, J biochem.136,643-649 (2004). The specific implementation method comprises the following steps:
1. the concentration of the protein samples to be detected is detected by using an ultraviolet absorption method, and the protein samples comprise control human collagen (Sigma, C7774), C2R1T, C2R4T, C2R5T and C2R10 protein samples. Specifically, the protein concentration was calculated using the empirical formula C (μ g/mL) 144X (a215-a225) to measure the uv absorption at 215nm and 225nm, respectively, taking care that detection is required with a215< 1.5. The method has the principle of measuring the characteristic absorption of peptide bonds under far ultraviolet light, is not influenced by the content of chromophore, has few interfering substances and simple and convenient operation, and is suitable for detecting human collagen and analogues thereof which do not develop color by Coomassie brilliant blue. (reference is made to Walker JM. the Protein Protocols Handbook, second edition. HumanaPress.43-45). After the protein concentration was determined, the concentration of all proteins to be tested was adjusted to 0.5mg/ml with PBS.
2. 100 μ l of each protein solution and a blank PBS solution control were added to a 96-well plate and allowed to stand at room temperature for 60 min.
3. Adding 10 into each hole5Well-cultured 3T3 cells (from the Physician of the university of Qinghua) were incubated at 37 ℃ for 60 min.
4. Each well was washed 4 times with PBS.
5. The absorbance of OD492nm was detected with LDH detection kit (Roche, 04744926001). According to the value of the blank control, the adherence rate of the cells can be calculated. The calculation formula is as follows: cell adherence rate ═ 100%/(positive well-blank well). The anchorage rate of the cells can reflect the activity of the collagen. The higher the activity of the protein, the better the external environment can be provided for the cells in a short time, and the cells are attached to the wall.
The results are shown in FIGS. 6 to 9.
The results of fig. 6 to 9 show that all four types of humanized type II collagen (i.e., C2R1T, C2R4T, C2R5T, and C2R10) have excellent adhesive activity compared to commercial human collagen. Wherein the adhesion effect of the C2R4T and the C2R10T is obviously better than that of the commercialized human collagen.
Example 3: preparation of HPV16, 18, 58 type pseudoviruses
Preparation of HPV types 16, 18, 58 HPV pseudoviruses (ref. reference: lu, l., Yang, x, Li, y, Jiang, s. (2013), chemical ly modified bone beta-lactigulin inhibitors human palladium infection. micro b. infection. mar 19.15(6-7):506-10, which and the references cited therein are incorporated by reference). Specifically, 293T cells (b)
Figure BDA0001968349440000221
CRL-3216) at 2X 105Inoculating to 35mm dishes at 37 ℃ with 5% CO2After 24h incubation, the HPV pseudoviral plasmids (addge, p16sheLL, #37320, HPV 16; p18sheLL, #37321, HPV 18; p58sheLL, #37324, HPV 58; 2.5. mu.g) were CO-transfected with reporter plasmids (pCLucf, addge, 2.5. mu.g) into 293T cells using the transfection reagent VigoFec (Vigorous) according to the manufacturer's instructions, followed by 5% CO transfection at 37 ℃ into 293T cells2Culturing overnight, replacing 20ml of new culture solution (2% FBS DMEM, Melen, MA0212) after 6 hours of transfection, digesting with 5ml of pancreatin (Melen, MB4375) after 48 hours until the cells are not attached to the wall, centrifuging (1000rpm, 3min) to remove the supernatant, lysing the cells with 10% Triton X-100, standing overnight at 37 ℃, centrifuging (12000rpm, 2min, 4 ℃) the next day, taking the supernatant, collecting the supernatant in a separate tube, and storing at-80 ℃ for later use.
Example 4: HPV infection inhibition assay
2.1 Experimental materials and methods
2.1.1 detection protein inhibits HPV16, 18 and 58 types of HPV pseudoviruses from infecting Hela cells: (
Figure BDA0001968349440000222
Method for the activity of CCL-2) as follows: the prepared protein sample C2R1T (C2R1T was prepared as described above, diluted to 20 μ M), was subjected to gradient dilution (20 μ M protein stock solution was prepared in the uppermost row, and sequentially diluted 2-fold, and the concentration ranges of optimum concentrations for different types were found from experiments, with an initial HPV16 type concentration of 6 μ M, an initial HPV18 type concentration of 12 μ M, and an initial HPV58 type concentration of 12 μ M), each concentration being 3 duplicate wells, mixed with each pseudovirus (prepared by adding 10 μ l of the virus stock solution prepared in example 3 to 10ml of DMEM) in equal volumes (50 μ l), and incubated at 25 ℃ for 30 minutes; a mixture of virus and sample to be tested (100. mu.l in total) was added to the pre-spread Hela cellsCell (1X 10)5) In 96-well plates at 37 ℃ with 5% CO2Culturing, and detecting the expression and enzyme activity of luciferase reporter gene of each well at 600nm by using an InfiniteM200Pro type microplate reader after 72 hours (using a determination kit Promega, E1500 according to the instructions of the manufacturer); cell wells (not infected with virus) were blank controls, and cell wells infected with only pseudovirus were positive controls, treated under the same conditions as the sample wells. The pseudovirus inhibition rate of each sample can be calculated by the following formula:
pseudoviral inhibition (%) - (average of positive control RLU-sample RLU)/(average of positive control RLU-average of blank RLU) 100
The median effect (entry inhibition) concentration (IC50) of each protein sample against each pseudovirus was calculated separately using CalcuSyn software.
FIGS. 10A-C show that C2R1T has inhibitory activity against HPV16, 18 and 58.
2.1.2 test of the polypeptides C2R1T, R1P1, R1P2, R1P3 and R1P4(C2R1T for laboratory expression purification. as described in example 1; R1P1, R1P2, R1P3 and R1P4 polypeptides are synthesized from Shanghaineqi peptide organisms) experiments for inhibiting HPV16 (polypeptides R1P1, R1P2, R1P3 and R1P4 starting at 913, 922, 908, 922. mu.M, triply graded dilution, respectively) the method differs from the above experimental method of 2.1.1 only in that the sample to be tested is a different polypeptide.
2.2 Experimental results and analysis
From the experimental results, the IC50 value of C2R1T against HPV16 type was 0.213. mu.M, the IC50 value against HPV18 type was 1.78. mu.M, and the IC50 value against HPV58 type was 1.27. mu.M.
From the experimental results, it was found that the polypeptides R1P1, R1P2 and R1P3 other than the polypeptide R1P4 had no activity of inhibiting HPV infection, and that the IC50 of R1P4 against HPV16 type was 237. mu.M, as shown in FIGS. 12A-D and Table 2.
Table 2: protein and polypeptide antiviral activity
Figure BDA0001968349440000231
Example 5C 2R1T anti-HPV mechanism.
In order to explore the action principle of C2R1T, an experiment is designed to discuss the action target of the C2R1T, and the Time-of-removal and ELISA experiments prove that C2R1T and HPV virus L1 protein are combined with each other to play an inhibitory activity.
3.1ELISA method as follows:
(1) see, e.g., chinese patent application No. 201410561005.1 or 201510393377.2 (incorporated herein by reference.) specifically, β -lactoglobulin is dissolved in phosphate buffer, ph8.5, to a final concentration of 20mg/ml, and then saturated 3-hydroxyphthalic anhydride is slowly added to this solution and shaken, the mixture is left at room temperature for 1 hour, dialyzed against ph7.4pbs, and then sterilized by filtration through a microfiltration membrane.
(2) Coating: C2R1T protein (5. mu.g/ml), JB01 (laboratory preparations), R1P4, and BSA (purchased from aMReSCO, 0332) were diluted in PBS (pH 9.6) at the same concentration (5. mu.g/ml) and added to ELISA assay plates at 50. mu.l per well overnight at 4 ℃.
(3) And (3) sealing: add 150. mu.l of 2% skim milk per well (0.05% Tween-20 in PBS), incubate for 2h at 37 ℃ and wash the plate 3 times with 0.05% PBS/Tween-20(PBST) wash.
(4) Antigen: blocked ELISA plates were loaded with 50. mu.l of HPV16L1 protein solution (5. mu.g/ml) (purchased from abcam, ab119880) per well, incubated for 1h at 37 ℃ and plates washed 3 times with PBST solution.
(5) A first antibody: mu.l of monoclonal antibody ab69 (1. mu.g/ml) (purchased from abcam, ab69) specifically recognizing HPV16L1 was added to each well, incubated at 37 ℃ for 1h, and the plates were washed 3 times with PBST solution.
(6) Secondary antibody: mu.l rabbit anti-mouse IgG-horseradish peroxidase (DACO, P0260) was added to each well, incubated for 1h, and the plates were washed 3 times.
(7) Color development and termination: ELISA developing solution (TMB, Biyunyan, P0209)50 μ l is added into each well, development is carried out for 5-10min, and then 50 μ l 2M H2SO4 is added to stop the development reaction. Data were obtained by detecting the OD450 of each well with a microplate reader (TECAN _ SNine M200 pro).
3.2Time-of-removal test method as follows:
100 μ l C2R1T (100 μ g/ml) was mixed with 1X 10 in DMEM medium (Melamine) previously cultured in 96-well cell culture plates5The Hela cells were incubated at 37 ℃ for 1 hour, and thenThe medium containing the sample was discarded and the cells were washed 3 times with DMEM (PBS) medium, and the washed cells were infected with pseudovirus (HPV16, 50. mu.l/well). A sample after adding an equal amount (50. mu.l/well) of pseudovirus to a sample well not washing C2R1T was used as a control (right column of FIG. 13). After 12 hours of incubation, the cells were collected by changing the medium (DMEM, 2% FBS, 200. mu.l) and after 72 hours of incubation, and the luciferase reporter gene expression was measured at 600nm using the kit (Promega, E1500) according to the manufacturer's instructions using an Infinite M200Pro microplate reader, and activities were compared using the rate of viral inhibition as a standard.
3.3 Virus inactivation assay method
50 μ l C2R1T (prepared as above, 1mg/ml diluted to final concentrations of 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.031mg/ml, respectively) and HPV16 virus (50 μ l stock solution prepared in example 3) were incubated on ice for 1h, virus was precipitated with 20% PEG8000 (national drug) and incubated on ice for 1h, centrifuged at 12000rpm for 30min, and the supernatant was discarded. Resuspend with 1ml PBS, reprecipitate with PEG8000 to remove impurities and C2R1T, and wash three times. The collected virus-infected cells after C2R1T action were incubated for 72h with the same amount of virus not acting on the drug as a positive control and a blank control without the sample and virus as a cell without the sample and luciferase reporter expression was detected using a kit (Promega, E1500) according to the manufacturer's instructions using an Infinite M200Pro microplate reader at 600nm, and the residual infection rate (%) (value [ (sample RLU-blank RLU average)/(positive control RLU average-blank RLU average) ] -100, higher value indicates a lower inactivation activity). Thus judging whether the product has inactivation activity.
3.4 results and analysis of the experiment
As shown in FIGS. 13 to 15, it was found from the results of the experiments that C2R1T acts at the entry stage of the virus, does not act on the target cells, and its binding to HPV L1 protein inhibits HPV infection and has no virus-inactivating activity.
Sequence listing
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ggaccggcag gagcaagagg aatacaggga ccgcagggac cgagaggaga taaaggtgaa 240
gcaggggagc cgggagaaag aggactgaaa ggacatagag gatttacagg agaagcagga 300
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ggaccgagag gggacaaagg agaagcggga gaaccgggag agagaggact gaagggacat 420
agagggttca cgggtgaagc cggagcacaa ggaccgatgg gtccgagcgg gccggcaggt 480
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Gly Pro Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly Arg Thr Gly
325 330 335
Pro Ala Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Gln Pro Gly Pro
340 345 350
Ala Gly Pro Pro Gly Pro Val Gly Pro Ala Gly Gly Pro Gly Phe Pro
355 360 365
Gly Ala Pro Gly Ala Lys Gly Glu Ala Gly Pro Thr Gly Ala Arg Gly
370 375 380
Pro Glu Gly Ala Gln Gly Pro Arg Gly Glu Pro Gly Thr Pro Gly Ser
385 390 395 400
Pro Gly Pro Ala Gly Ala Ser Gly Asn Pro Gly Thr Asp Gly Ile Pro
405 410 415
Gly Ala Lys Gly Ser Ala Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly
420 425 430
Phe Pro Gly Pro Arg Gly Pro Pro Gly Pro Gln Gly Ala Thr Gly Pro
435 440 445
Leu Gly Pro Lys Gly Gln Thr Gly Glu Pro Gly Ile Ala Gly Phe Lys
450 455 460
Gly Glu Gln Gly Pro Lys Gly Glu Pro Gly Pro Ala Gly Pro Gln Gly
465 470 475 480
Ala Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu
485 490 495
Pro Gly Gly Val Gly Pro Ile Gly Pro Pro Gly Glu Arg Gly Ala Pro
500 505 510
Gly Asn Arg Gly Phe Pro Gly Gln Asp Gly Leu Ala Gly Pro Lys Gly
515 520 525
Ala Pro Gly Glu Arg Gly Pro Ser Gly Leu Ala Gly Pro Lys Gly Ala
530 535 540
Asn Gly Asp Pro Gly Arg Pro Gly Glu Pro Gly Leu Pro Gly Ala Arg
545 550 555 560
Gly Leu Thr Gly Arg Pro Gly Asp Ala Gly Pro Gln Gly Lys Val Gly
565 570 575
Pro Ser Gly Ala Pro Gly Glu Asp Gly Arg Pro Gly Pro Pro Gly Pro
580 585 590
Gln Gly Ala Arg Gly Gln Pro Gly Val Met Gly Phe Pro Gly Pro Lys
595 600 605
Gly Ala Asn Gly Glu Pro Gly Lys Ala Gly Glu Lys Gly Leu Pro Gly
610 615 620
Ala Pro Gly Leu Arg Gly Leu Pro Gly Lys Asp Gly Glu Thr Gly Ala
625 630 635 640
Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln
645 650 655
Gly Ala Pro Gly Pro Ser Gly Phe Gln Gly Leu Pro Gly Pro Pro Gly
660 665 670
Pro Pro Gly Glu Gly Gly Lys Pro Gly Asp Gln Gly Val Pro Gly Glu
675 680 685
Ala Gly Ala Pro Gly Leu Val Gly Pro Arg Gly Glu Arg Gly Phe Pro
690 695 700
Gly Glu Arg Gly Ser Pro Gly Ala Gln Gly Leu Gln Gly Pro Arg Gly
705 710 715 720
Leu Pro Gly Thr Pro Gly Thr Asp Gly Pro Lys Gly Ala Ser Gly Pro
725 730 735
Ala Gly Pro Pro Gly Ala Gln Gly Pro Pro Gly Leu Gln Gly Met Pro
740 745 750
Gly Glu Arg Gly Ala Ala Gly Ile Ala Gly Pro Lys Gly Asp Arg Gly
755 760 765
Asp Val Gly Glu Lys Gly Pro Glu Gly Ala Pro Gly Lys Asp Gly Gly
770 775 780
Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Asn
785 790 795 800
Gly Glu Lys Gly Glu Val Gly Pro Pro Gly Pro Ala Gly Ser Ala Gly
805 810 815
Ala Arg Gly Ala Pro Gly Glu Arg Gly Glu Thr Gly Pro Pro Gly Pro
820 825 830
Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys
835 840 845
Gly Glu Gln Gly Glu Ala Gly Gln Lys Gly Asp Ala Gly Ala Pro Gly
850 855 860
Pro Gln Gly Pro Ser Gly Ala Pro Gly Pro Gln Gly Pro Thr Gly Val
865 870 875 880
Thr Gly Pro Lys Gly Ala Arg Gly Ala Gln Gly Pro Pro Gly Ala Thr
885 890 895
Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Ser Asn Gly
900 905 910
Asn Pro Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Lys Asp Gly Pro
915 920 925
Lys Gly Ala Arg Gly Asp Ser Gly Pro Pro Gly Arg Ala Gly Glu Pro
930 935 940
Gly Leu Gln Gly Pro Ala Gly Pro Pro Gly Glu Lys Gly Glu Pro Gly
945 950 955 960
Asp Asp Gly Pro Ser Gly Ala Glu Gly Pro Pro Gly Pro Gln Gly Leu
965 970 975
Ala Gly Gln Arg Gly Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg
980 985 990
Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly
995 1000 1005
Ala Pro Gly Ala Ser Gly Asp Arg Gly Pro Pro Gly Pro Val Gly
1010 1015 1020
Pro Pro Gly Leu Thr Gly Pro Ala Gly Glu Pro Gly Arg Glu Gly
1025 1030 1035
Ser Pro Gly Ala Asp Gly Pro Pro Gly Arg Asp Gly Ala Ala Gly
1040 1045 1050
Val Lys Gly Asp Arg Gly Glu Thr Gly Ala Val Gly Ala Pro Gly
1055 1060 1065
Ala Pro Gly Pro Pro Gly Ser Pro Gly Pro Ala Gly Pro Thr Gly
1070 1075 1080
Lys Gln Gly Asp Arg Gly Glu Ala Gly Ala Gln Gly Pro Met Gly
1085 1090 1095
Pro Ser Gly Pro Ala Gly Ala Arg Gly Ile Gln Gly Pro Gln Gly
1100 1105 1110
Pro Arg Gly Asp Lys Gly Glu Ala Gly Glu Pro Gly Glu Arg Gly
1115 1120 1125
Leu Lys Gly His Arg Gly Phe Thr Gly Leu Gln Gly Leu Pro Gly
1130 1135 1140
Pro Pro Gly Pro Ser Gly Asp Gln Gly Ala Ser Gly Pro Ala Gly
1145 1150 1155
Pro Ser Gly Pro Arg Gly Pro Pro Gly Pro Val Gly Pro Ser Gly
1160 1165 1170
Lys Asp Gly Ala Asn Gly Ile Pro Gly Pro Ile Gly Pro Pro Gly
1175 1180 1185
Pro Arg Gly Arg Ser Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly
1190 1195 1200
Asn Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Gly Ile
1205 1210 1215
Asp Met Ser Ala Phe Ala Gly Leu Gly Pro Arg Glu Lys Gly Pro
1220 1225 1230
Asp Pro Leu Gln Tyr Met Arg Ala Asp Gln Ala Ala Gly Gly Leu
1235 1240 1245
Arg Gln His Asp Ala GluVal Asp Ala Thr Leu Lys Ser Leu Asn
1250 1255 1260
Asn Gln Ile Glu Ser Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn
1265 1270 1275
Pro Ala Arg Thr Cys Arg Asp Leu Lys Leu Cys His Pro Glu Trp
1280 1285 1290
Lys Ser Gly Asp Tyr Trp Ile Asp Pro Asn Gln Gly Cys Thr Leu
1295 1300 1305
Asp Ala Met Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys
1310 1315 1320
Val Tyr Pro Asn Pro Ala Asn Val Pro Lys Lys Asn Trp Trp Ser
1325 1330 1335
Ser Lys Ser Lys Glu Lys Lys His Ile Trp Phe Gly Glu Thr Ile
1340 1345 1350
Asn Gly Gly Phe His Phe Ser Tyr Gly Asp Asp Asn Leu Ala Pro
1355 1360 1365
Asn Thr Ala Asn Val Gln Met Thr Phe Leu Arg Leu Leu Ser Thr
1370 1375 1380
Glu Gly Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala
1385 1390 1395
Tyr Leu Asp Glu Ala Ala Gly Asn Leu Lys Lys Ala Leu Leu Ile
1400 14051410
Gln Gly Ser Asn Asp Val Glu Ile Arg Ala Glu Gly Asn Ser Arg
1415 1420 1425
Phe Thr Tyr Thr Ala Leu Lys Asp Gly Cys Thr Lys His Thr Gly
1430 1435 1440
Lys Trp Gly Lys Thr Val Ile Glu Tyr Arg Ser Gln Lys Thr Ser
1445 1450 1455
Arg Leu Pro Ile Ile Asp Ile Ala Pro Met Asp Ile Gly Gly Pro
1460 1465 1470
Glu Gln Glu Phe Gly Val Asp Ile Gly Pro Val Cys Phe Leu
1475 1480 1485
<210>14
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223> peptide fragment
<400>14
Gly Pro Pro Gly Pro Cys Cys Gly Gly Gly
1 5 10
<210>15
<211>30
<212>PRT
<213> Artificial sequence
<220>
<223>R1P1
<400>15
Gly Lys Asp Gly Pro Lys Gly Ala Arg Gly Asp Ser Gly Pro Pro Gly
1 5 10 15
Arg Ala Gly Glu Pro Gly Leu Gln Gly Pro Ala Gly Pro Pro
20 25 30
<210>16
<211>30
<212>PRT
<213> Artificial sequence
<220>
<223>R1P2
<400>16
Gly Ala Arg Gly Asp Ser Gly Pro Pro Gly Arg Ala Gly Glu Pro Gly
1 5 10 15
Leu Gln Gly Pro Ala Gly Pro Pro Gly Glu Lys Gly Glu Pro
20 25 30
<210>17
<211>30
<212>PRT
<213> Artificial sequence
<220>
<223>R1P3
<400>17
Gly Pro Pro Gly Arg Ala Gly Glu Pro Gly Leu Gln Gly Pro Ala Gly
1 5 10 15
Pro Pro Gly Glu Lys Gly Glu Pro Gly Asp Asp Gly Pro Ser
20 25 30
<210>18
<211>30
<212>PRT
<213> Artificial sequence
<220>
<223>R1P4
<400>18
Gly Glu Pro Gly Leu Gln Gly Pro Ala Gly Pro Pro Gly Glu Lys Gly
1 5 10 15
Glu Pro Gly Asp Asp Gly Pro Ser Gly Ala Glu Gly Pro Pro
20 25 30

Claims (21)

1. The polypeptide consists of an amino acid sequence shown by SEQ ID number 5 or SEQ ID NO. 18.
2. A polynucleotide encoding the polypeptide of claim 1.
3. The polynucleotide according to claim 2, which consists of the sequence of SEQ ID number 6.
4. An expression vector comprising the polynucleotide of claim 2 or 3.
5. A host cell comprising the expression vector of claim 4.
6. The host cell of claim 5, wherein the host cell is E.
7. A method for producing a polypeptide according to claim 1, comprising:
(1) culturing the host cell of claim 5 or 6 in a production medium and producing the polypeptide; and
(2) the polypeptide is harvested and purified.
8. The method of claim 7, further comprising cleaving the polypeptide with an enzyme.
9. A composition comprising the polypeptide of claim 1.
10. The composition of claim 9, wherein the composition is a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic, or a nutraceutical.
11. The composition of claim 10, wherein the pharmaceutical composition is an external preparation.
12. The composition of claim 11, wherein the pharmaceutical composition is an external spread formulation.
13. The composition of claim 12, wherein the pharmaceutical composition is a topical gel or a topical infiltrant formulation.
14. The composition of claim 13, wherein the topical gel further comprises a pharmaceutically acceptable carrier vehicle and the topical infiltration formulation further comprises a sterile medical cotton ball having the polypeptide of claim 1.
15. Use of the polypeptide of claim 1, the polynucleotide of claim 2 or 3, the expression vector of claim 4, the host cell of claim 5 or 6, or the composition of any one of claims 9 to 14 for the preparation of a medical device, a tissue engineering product, a cosmetic product or a nutraceutical product.
16. Use of a polypeptide according to claim 1, a polynucleotide according to claim 2 or 3, an expression vector according to claim 4, a host cell according to claim 5 or 6 or a composition according to any one of claims 9 to 14 in the manufacture of a product for the prevention or treatment of an HPV infection or a disease caused by an HPV infection.
17. The use of claim 16, wherein the HPV infection is positive for HPV detection and the disease caused by HPV infection is a disease caused by persistent infection with HPV.
18. The use according to claim 17, wherein the disease caused by HPV persistent infection is cervical neoplasia, condyloma acuminatum or cervical cancer.
19. The use according to claim 16, wherein the HPV is HPV type 16, 18 and/or 58.
20. Use of the polypeptide of claim 1, the polynucleotide of claim 2 or 3, the expression vector of claim 4, the host cell of claim 5 or 6, or the composition of any one of claims 9-14 to inhibit HPV or bind HPV L1 protein in vitro.
21. The use according to claim 20, wherein the HPV is HPV type 16, 18 and/or 58.
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