WO2021013063A1 - Chimeric human papillomavirus type 16 l1 protein - Google Patents

Abstract

A chimeric human papillomavirus type 16 L1 protein, a polynucleotide encoding same, an HPV type 16 virus-like particle, and a preparation method therefor. The chimeric human papillomavirus type 16 L1 protein comprises an N-terminal fragment derived from an HPV type 16 L1 protein, the N-terminal fragment retaining the immunogenicity of the HPV type 16 L1 protein; and a C-terminal fragment derived from an L1 protein of a second type of papillomavirus having better expression and solubility characteristics compared to L1 proteins of other types of papillomavirus. The chimeric HPV type 16 L1 protein has the immunogenicity of the HPV type 16 L1 protein. The chimeric human papillomavirus type 16 L1 protein has a high expression level and high solubility and can be used in large-scale production of a vaccine.

Description

Chimeric human papillomavirus type 16 L1 protein Technical field

The present invention relates to human papillomavirus (HPV) L1 protein and polynucleotide encoding the protein, and also relates to HPV virus-like particles and a preparation method thereof.

Background technique

Papilloma virus (PV) belongs to the family of papillomaviruses (Papillomaviridae) and can cause papilloma in humans, cattle, dogs, rabbits, etc. Its member human papillomavirus (Human Papillomavirus, HPV) is a non-enveloped DNA virus. The genome of the virus is a double-stranded closed-loop DNA with a size of about 7.2-8 kb and 8 open reading frames, which can be divided into three regions according to their functions: (1) early region (E), about 4.5 kb, encoding E1, E2 E4-E7 are 6 non-structural proteins related to virus replication, transcription and transformation; (2) Late region (L), about 2.5kb, encoding major capsid protein L1 and minor capsid protein L2; (3) long The regulatory region (LCR), located between the end of the L region and the beginning of the E region, is about 800-900 bp in length, does not encode any protein, but has DNA replication and expression regulatory elements.

L1 and L2 proteins are synthesized in the middle and late stages of the HPV infection cycle. The L1 protein is the main capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is a minor capsid protein. 72 L1 protein pentamers constitute the outer shell of icosahedral HPV virus particles (45-55nm in diameter), which wraps the closed-loop double-stranded DNA. The L2 protein is located inside the L1 protein (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, X.S., R.L. Garcea, Mol. Cell. 5(3): 557-567, 2000).

The ORF of L1 protein is the most conserved gene in PV genome and can be used to identify new PV types. If the complete genome is cloned, and the DNA sequence of the L1 ORF differs by more than 10% from the closest known PV type, it is considered to have isolated a new PV type. Differences between 2% and 10% homology are defined as different subtypes, and differences less than 2% are defined as different variants of the same subtype (E.-M.de Villiers et al./Virology 324(2004) 17- 27).

In the later stage of HPV infection, the newly synthesized L1 protein in the cytoplasm is transported to the terminally differentiated keratin cell nucleus. Together with the L2 protein, the copied HPV genomic DNA is packaged to form an infectious virus (Nelson, LM, et al. 2002. Nuclear import strategies of high risk HPV16 L1 major capsid protein. J.Biol.Chem.277:23958-23964). This indicates that the nuclear introduction of L1 protein plays a very important role in HPV infection and production. The ability of the virus to enter the nucleus is determined by the nuclear localization signal (NLS) at the C-terminal of the HPV L1 protein. A feature of the nuclear localization signal is that it is rich in basic amino acids (Garcia-Bustos, J., et al. 1991. Nuclear protein localization. Biochimica et al. Biophysica Acta 1071:83-101).

15 high-risk (HR) HPV types can cause cancer of the cervix, anus, penis, vagina, vulva, and oropharynx. Among them, HPV-16 and HPV-18 are the most common causes of cancer so far, accounting for about 70% of cervical cancer, and the rest are other HR-HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82) caused. HPV-16 accounts for about 95% of HPV-positive oropharyngeal carcinomas (OPCs). The persistent low-risk genotypes HPV-6 and HPV-11 cause most anogenital warts and respiratory papilloma, but they are rarely associated with cancer (Human Papillomavirus in Cervical Cancer and Oropharyngeal Cancer: One Cause, Two Diseases Tara A. Berman and John T. Schiller, PhD2 Cancer 2017; 123:2219-29).

Using vaccinia virus, baculovirus or yeast system to recombinantly express L1 protein, the L1 protein can self-assemble to form a virus-like particle (VLP), which contains about 72 L1 proteins, which is similar to the virion shell. VLP has no indications. VLP can induce neutralizing antibodies in vaccinated animals and protect laboratory animals from subsequent attacks by infectious viruses. Therefore, VLP seems to be an excellent candidate for papillomavirus vaccine (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, XS, RL Garcea, Mol. Cell. 5(3): 557-567, 2000).

Glaxo's

It is a bivalent recombinant HPV vaccine. It contains the HPV 16 type recombinant L1 protein and the HPV 18 type recombinant L1 protein obtained by the recombinant baculovirus expression vector system in the insect cells of Trichoplusia ni. L1 protein self-assembles into virus-like particles, which are used to prevent cervical cancer caused by HPV types 16 and 18 in women aged 9-25, grade 2 or 3 cervical intraepithelial neoplasia and adenocarcinoma in situ, and grade 1 cervical cancer Intraepithelial neoplasia (carcinogenic) (https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM186981.pdf).

It is a human papillomavirus quadrivalent (type 6, 11, 16 and 18) recombinant vaccine produced by Merck. It is used in girls and women aged 9-26 to prevent cervical cancer from genital warts (condyloma acuminatum) and caused by HPV. Types 6, 11, 16, and 18 cause precancerous or hyperplastic abnormalities; and boys and men aged 9-26 are used to prevent anal cancer, genital warts (condyloma acuminatum), and HPV types 6, 11, 16, 18 Precancerous or dysplastic lesions (https://www.fda.gov/vaccines-blood-biologics/vaccines/gardasil).

It is a 9-valent recombinant human papilloma virus vaccine produced by Merck, which contains virus-like particles of HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 L1 protein, which is fermented by Saccharomyces cerevisiae Production, self-assembly as VLP. For girls and women aged 9-45 for the prevention of cervical cancer, vulvar cancer, vaginal cancer and anal cancer caused by HPV 16, 18, 31, 33, 45, 52 and 58, and genital warts (sharp) caused by HPV 6 and 11 Condyloma) and precancerous or hyperplastic abnormalities caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58; and boys and men aged 9-45 for the prevention of 16, 18, 31, Anal cancer caused by types 33, 45, 52 and 58, genital warts (condyloma acuminatum) caused by HPV 6 and 11, and precancerous stages caused by HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 Or dysplastic lesions (https://www.fda.gov/vaccines-blood-biologics/vaccines/gardasil-9).

The instructions stated that HPV 16 and 18 are the cause of about 70% of cervical cancers, and the remaining 20% of cases are attributed to types 31, 33, 45, 52, and 58.

It can prevent 90% of cervical cancers (https://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedPro ducts/ucm426445.htm).

The key factor in HPV vaccine development is that virus-like particles can be produced in large quantities. At present, the more common systems for producing virus-like particles are mainly divided into eukaryotic expression systems and prokaryotic expression systems.

Commonly used eukaryotic expression systems include poxvirus expression system, insect baculovirus expression system, and yeast expression system. The HPV L1 protein expressed in the eukaryotic expression system has less natural conformation damage and can assemble spontaneously to form virus-like particles, but the yield is low. The prokaryotic expression system is mainly Escherichia coli expression system, with high yield but mostly in the form of inclusion bodies, which is not conducive to purification and the production process is complicated.

Therefore, there is still a need to obtain high-yield HPV virus-like particles in this field.

Summary of the invention

In one aspect, the present invention provides a chimeric human papillomavirus (HPV) type 16 L1 protein, comprising a. N-terminal fragment derived from HPV type 16 L1 protein from its N-terminal to C-terminal direction, said N The terminal fragment maintains the immunogenicity of the HPV type 16 L1 protein; and b. The C-terminal fragment derived from the L1 protein of the second type papillomavirus, which has a higher level of Other L1 protein expression and solubility are better; the chimeric HPV type 16 L1 protein has the immunogenicity of HPV type 16 L1 protein.

In another aspect, the present invention provides a HPV type 16 virus-like particle, which comprises a chimeric HPV type 16 L1 protein.

In another aspect, the present invention provides an immunogenic composition for preventing HPV-related diseases or infections, which comprises HPV type 16 virus-like particles and an adjuvant.

In another aspect, the present invention provides an isolated polynucleotide encoding a chimeric HPV type 16 L1 protein.

In another aspect, the present invention provides a vector comprising a polynucleotide encoding a chimeric HPV type 16 L1 protein.

In another aspect, the present invention provides a baculovirus comprising a polynucleotide encoding a chimeric HPV type 16 L1 protein.

In another aspect, the present invention provides a host cell comprising the polynucleotide, vector, or baculovirus as described above.

In another aspect, the present invention provides a method for preparing HPV type 16 virus-like particles, which comprises culturing the host cell as described above to express the chimeric HPV type 16 L1 protein and assemble it into virus-like particles; and purification The HPV type 16 virus-like particles.

Description of the drawings

Figure 1 HPV 16L1: 33C L1 protein expression. M: Marker; L: cell lysate; E-S: supernatant collected after centrifugation of the lysate.

Figure 2 Transmission electron microscope observation of HPV 16L1: 33C virus-like particles.

Figure 3 Expression of HPV16L1 (1-474) truncated at the C-terminal. M: Marker; L: cell lysate; E-S: supernatant collected after centrifugation of the lysate.

Detailed ways

In one aspect, the present invention provides a chimeric human papillomavirus (HPV) type 16 L1 protein, from its N-terminal to C-terminal direction comprising: a. an N-terminal fragment derived from HPV type 16 L1 protein, said The N-terminal fragment maintains the immunogenicity of the HPV type 16 L1 protein; and b. The C-terminal fragment derived from the L1 protein of the second type papillomavirus, which has a comparison with other types of papillomavirus L1 protein Types of L1 protein expression and solubility are better; wherein the chimeric HPV type 16 L1 protein has the immunogenicity of HPV type 16 L1 protein.

In one embodiment, the N-terminal fragment is a fragment obtained by truncating the C-terminus of the natural sequence of HPV type 16 L1 protein to any amino acid position in its α5 region, and is at least 98% identical to it. Sexual fragment; the C-terminal fragment is a fragment obtained by truncating the N-terminus of the natural sequence of the second type papillomavirus L1 protein to any amino acid position in its α5 region, and the fragment is further mutated , Deletions and/or additions resulting from functional variants.

In another embodiment, the N-terminal fragment and the fragment obtained by truncating the C-terminal of the natural sequence of HPV type 16 L1 protein to any amino acid position in its α5 region have at least 98.5%, 99%, 99.5% or 100% identity.

In another embodiment, the C-terminal fragment contains one or more nuclear localization sequences.

In one embodiment, the L1 protein of the second type of papillomavirus is selected from HPV type 1, type 2, type 3, type 4, type 6, type 7, type 10, type 11, type 13, type 16, and type 18. Type, 22 type, 26 type, 28 type, 31 type, 32 type, 33 type, 35 type, 39 type, 42 type, 44 type, 45 type, 51 type, 52 type, 53 type, 56 type, 58 type, Type 59, Type 60, Type 63, Type 66, Type 68, Type 73 or Type 82 L1 protein;

Preferably, the L1 protein of the second type of papillomavirus is selected from HPV type 16, type 28, type 33, type 59, or type 68 L1 protein;

More preferably, the L1 protein of the second type of papillomavirus is selected from HPV type 33 or HPV type 59 L1 protein.

In one embodiment, the L1 protein of the second type of papillomavirus is HPV type 33 L1 protein, and the C-terminal fragment is SEQ ID No: 2; or a fragment of m amino acids in length, preferably covering SEQ ID No: 2 is a fragment of amino acid at position 1-m; where m is an integer from 8-26.

In one embodiment, the C-terminal fragment of HPV type 33 L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV type 33 L1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV type 16 L1 protein comprises one or more C-terminal fragments of HPV type 33 L1 protein. The C-terminal fragments of the multiple HPV type 33 L1 proteins may be the same or different. In one embodiment, the amino acid sequence (KR) of amino acid number 7-8 of SEQ ID No: 2 and the amino acid sequence of amino acid sequence number 20-23 (KRKK) are the nuclear localization sequence of the C-terminal fragment of HPV 33 type L1 protein .

In another embodiment, the second type of papillomavirus L1 protein is HPV59 type L1 protein, and the C-terminal fragment is SEQ ID No: 13; or a fragment of n amino acids in length, preferably covering SEQ ID No: 13 is a fragment of amino acids 1-n; where n is an integer from 16 to 38.

In one embodiment, the C-terminal fragment of HPV type 59 L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV type 59 L1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV type 16 L1 protein comprises one or more C-terminal fragments of HPV type 59 L1 protein. The C-terminal fragments of the multiple HPV type 59 L1 proteins may be the same or different. In one embodiment, the amino acid sequence (RKR) of amino acid number 14-16 of SEQ ID No: 13 and the amino acid sequence of amino acid sequence number 28-34 (KRVKRRK) are the nuclear localization sequence of the C-terminal fragment of HPV type 59 L1 protein .

In one embodiment, the chimeric HPV type 16 L1 protein includes both the C-terminal fragment of HPV type 33 L1 protein and the C-terminal fragment of HPV type 59 L1 protein.

In one embodiment, the N-terminal fragment and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No:1 to any amino acid position in its α5 region have 98%, 98.5%, and 99% , 99.5% or 100% identity.

In one embodiment, the C-terminus of the N-terminal fragment and the N-terminus of the C-terminal fragment are directly connected or connected via a linker.

The linker does not affect the immunogenicity of the N-terminal fragment, and does not affect the expression or solubility of the protein. In one embodiment, the N-terminal fragment and the C-terminal fragment are connected by a linker consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In one embodiment, the linker is an artificial sequence. In another embodiment, the linker is a sequence naturally occurring in the HPV L1 protein. In one embodiment, the linker may be a partial sequence of HPV type 16 L1 protein. In another embodiment, the linker may be a partial sequence of HPV type 33 L1 protein. In another embodiment, the linker may be a partial sequence of HPV type 59 L1 protein.

In one embodiment, when the C-terminus of the N-terminal fragment is connected to the N-terminus of the C-terminal fragment, the following continuous amino acid sequence exists within the range of plus and minus 4 amino acid positions of the connection point: RKFL; preferably Ground, the following continuous amino acid sequence exists in the range of plus or minus 6 amino acid positions of the connection point: LGRKFL.

In one embodiment, the chimeric HPV type 16 L1 protein has 98%, 98.5%, 99%, 99.5% or 100% identity with SEQ ID No: 3.

In another aspect, the present invention provides a HPV type 16 virus-like particle, which comprises the chimeric HPV type 16 L1 protein as described above. In one embodiment, the HPV type 16 virus-like particle is an icosahedron composed of 72 pentamers of the chimeric HPV type 16 L1 protein. In one embodiment, HPV type 16 virus-like particles have correctly formed disulfide bonds and therefore have a good natural conformation. In one embodiment, HPV type 16 virus-like particles self-assemble in the in vivo expression system.

In one aspect, the present invention provides an immunogenic composition for preventing HPV-related diseases or infections, which comprises the HPV type 16 virus-like particles as described above and an adjuvant. The prevention can be considered a treatment, and the two can be used interchangeably.

In one aspect, the above immunogenic composition is administered to a subject. In one embodiment, the subject is a human.

In one aspect, the present invention provides an isolated polynucleotide encoding the chimeric HPV type 16 L1 protein as described above. In one embodiment, the polynucleotide is a polynucleotide that has been codon optimized for different expression systems. In one embodiment, the polynucleotide is a polynucleotide that has been codon optimized for the insect baculovirus expression system.

In one aspect, the present invention provides an isolated polynucleotide having the sequence shown in SEQ ID No: 4.

In one aspect, the present invention provides a vector comprising the polynucleotide as described above. In one embodiment, the vector is a baculovirus vector. In one embodiment, the vector may be a transfer vector used in a baculovirus expression system. In another embodiment, the vector may be an expression vector used in a baculovirus expression system. In another embodiment, the vector may be a recombined vector used in a baculovirus expression system.

In one aspect, the present invention provides a baculovirus comprising the polynucleotide as described above.

In one aspect, the present invention provides a host cell comprising the polynucleotide, vector, or baculovirus as described above. In one embodiment, the host cell is an insect cell. Preferably, the insect cell is selected from Sf9 cell, Sf21 cell, Hi5 cell and S2 cell.

In one aspect, the present invention provides a method for preparing HPV type 16 virus-like particles as described above, which comprises: culturing a host cell as described above to express the chimeric HPV type 16 L1 protein and assembling Into virus-like particles; and purifying the HPV 16 virus-like particles.

In one embodiment, the host cell is an insect cell. In one embodiment, the host cell is a Hi5 cell. In one embodiment, the chimeric HPV type 16 L1 protein self-assembles into HPV type 16 virus-like particles in the host cell. In one embodiment, the chimeric HPV type 16 L1 protein self-assembles into HPV type 16 virus-like particles in the host cell, which has a two-dimensional structure composed of 72 pentamers of the chimeric HPV type 16 L1 protein. Decahedron. In one embodiment, HPV type 16 virus-like particles have correctly formed disulfide bonds and thus have a good natural conformation.

In one embodiment, the purification uses cation exchange chromatography. In one embodiment, strong cation exchange chromatography is used for purification. In another embodiment, the purification uses weak cation exchange chromatography. In one embodiment, the purification uses a combination of multiple cation exchange chromatography. In one embodiment, HS strong cation exchange chromatography is used for purification. In another embodiment, MMA ion exchange chromatography is used for purification. In another embodiment, HS-MMA two-step chromatography is used for purification.

The papillomavirus L1 protein expressed by the eukaryotic expression system can spontaneously assemble into virus-like particles, but has the disadvantage of low expression and difficult mass production.

The sequence of the L1 protein of each type of HPV can be conveniently obtained from https://www.uniprot.org. Each type of HPV L1 can be derived from different strains, so its amino acid sequence has multiple versions, and any one version of the natural sequence can be used in the present invention. In the conception and design process of the present invention, a certain The sequence of the HPV L1 protein of a given type may be different from the sequence used in the examples, but this difference does not affect the judgment and conclusion of the inventor.

Those skilled in the art generally believe that the C-terminus of the L1 protein does not contain a main neutralizing epitope, so they try to increase the expression level by truncating the C-terminus of the HPV L1 protein. For example, in the U.S. Patent No. 6,361,778B1 of Glaxo, the C-terminus of HPV16L1 protein Truncating 1-34 amino acids, preferably 26 amino acids, declares that the yield of VLP is increased many times, preferably at least 10 times, especially about 10 to 100 times. Inspired by this, the inventors tried to shorten the C-terminus of HPV16 type L1 by 31 amino acids and named it HPV16L1 (1-474). However, its protein expression is high but the protein solubility is poor, and it is difficult to extract and purify (see comparative example).

The poor solubility of the protein caused by this truncation may be caused by the defect of the C-terminal nuclear localization sequence, and the present invention is not limited to this speculation. In the process of research and production, the inventor found that HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1 protein and HPV type 68 L1 protein are compared with other types of L1 protein expression and solubility Better, inspired by this, the inventor replaced the C-terminus of the HPV type that was difficult to extract or had a low expression level with the C-terminus of the L1 protein with better expression and solubility. That is, the inventors constructed such a chimeric protein which contains the N-terminal fragment derived from the first type of papillomavirus L1 protein (such as HPV L1 protein) and the second type of papillae from the N-terminal to the C-terminal direction. The C-terminal fragment of oncovirus L1 protein (such as HPV L1 protein). The former provides the immunogenicity of the first type of papillomavirus (such as HPV), and the latter provides the characteristics of better expression and solubility. The two can be connected directly or through a joint.

In order to maintain the immunogenicity of the first type of HPV L1 protein and ensure that it can form a VLP, the inventors determined the appropriate length of the N-terminal fragment of the HPV L1 protein. The following reports involve epitope studies of common HPV subtypes:

Sunanda Baidya et al reported that the L1 protein epitopes may be used as design tools 48EEYDLQFIFQLCKITLTA65,45RHGEEYDLQFIFQLCKITLTA65,63LPDPNKF69,79PETQRLVWAC88,36PVPGQYDA43,77YNPETQRLVWAC88,188DTGYGAMD195,36PVPGQYDATK45,45KQDIPKVSAYQYRVFRV61,130RDNVSVDYKQTQLCI144and49YSRHVEEY DLQFIF62 HPV16 and 18 vaccine (see Epitope design of L1 protein for Vaccine production against Human Papilloma Virus types 16 and 18, Bioinformation 13(3): 86-93 March 2017, all incorporated herein by reference).

Katharina Slupetzky et al. reported that the regions near aa 282-286 and 351-355 of HPV-16 contribute to neutralizing epitopes, and the latter are immunodominant sites (see Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops, Journal of General Virology (2001), 82, 2799-2804, fully incorporated herein by reference).

Brooke Bishop et al. prepared the following 3 variants of HPV11, 16, 18 and 35L1 proteins: 9 amino acid deletions at the N-terminus, α4 (corresponding to amino acid residues 404-436 of HPV16) deletion, and 31 C-terminus Amino acid deletion, it is reported that the former two cannot be assembled into VLP, but the latter is not reported

(Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins UNDERSTANDING THE SPECIFICITY OF NEUTRALIZING MONOCLONAL ANTIBODIES, The Journal of Biological Chemistry, 282, 31803-31811). This article is incorporated by reference in its entirety. The loop regions of the α helix and β sheet of each type of HPV L1 protein can be easily determined by sequence analysis software commonly used in this field. Among them, the α-helix region includes α1, α2, α3, α4, and α5 regions.

Inventor's comments on 14 types (type 6, type 11, type 16, type 18, type 31, type 33, type 35, type 39, type 45, type 51, type 52, type 56, type 58 and type 59) Align the sequence of the HPV L1 protein, and then perform the sequence alignment according to the above-cited literature (Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins UNDERSTANDING THE SPECIFICITY OF NEUTRALIZING MONOCLONAL ANTIBODIES, Theological 282, Second-level Chemist 282, The Journal 318) The structure prediction and the results are shown below, where the part between the downward arrows corresponds to the region that was missing for the preparation of the variant in this document.

In addition to the method of sequence comparison used by the inventors, the protein secondary structure prediction software that can be used for prediction includes but is not limited to:

1.JPred: http://www.compbio.dundee.ac.uk/jpred/index.html

2.ProtPredicct: http://predictprotein.org

3.PsiPred: http://bioinf.cs.ucl.ac.uk/psipred

4.SCRATCH-1D: http://download.igb.uci.edu

5. Nnpredict: http://www.cmpharm.ucsf.edu/～nomi/nnpredict

6.Predictprotein: http://www.embl-heidelberg.de/predictprotein/SOPMA http://www.ibcp.fr/predict.html

7. SSPRED: http://www.embl-heidelberg.de/sspred/ssprd_info.html.

In one embodiment of the present invention, the inventors determined the length of the N-terminal fragment of the L1 protein derived from the first type of HPV in the following manner: truncated the natural sequence of the L1 protein in its α5 region and its vicinity, and kept the length from The sequence from the N-terminal to the C-terminal newly generated in the α5 region. Such a truncated sequence can ensure that it has the immunogenicity of this type and can form a VLP.

The N-terminal fragment derived from the HPV L1 protein of the first type can be further modified to ensure that it has the immunogenicity of this type and can form a VLP.

The inventors determined the length of the C-terminal fragment derived from the second type of HPV L1 protein in the following manner. The natural sequence of L1 protein was truncated in its α5 region and its vicinity, and the newly generated N-terminal to C-terminal sequence from its α5 region was retained. Such a truncated sequence does not have a main neutralizing epitope and does not interfere with the immunogenicity of the chimeric protein formed.

The C-terminal fragment derived from the second type of HPV L1 protein may be further mutated, deleted and/or added, preferably retaining at least one nuclear localization sequence. Yang et al. predicted the nuclear localization sequence of 107 HPV subtypes (Yang et al. Predicting the nuclear localization signals of 107 types of HPV L1 proteins by bioinformatic analysis.Geno.Prot.Bioinfo.Vol. 4 No. 1 2006 by reference All are incorporated herein), the nuclear localization sequence of each type of HPV L1 protein can be easily determined by sequence analysis software commonly used in this field.

The ligation of the aforementioned N-terminal fragment and the C-terminal fragment occurs at the newly generated C-terminus of the former and the newly generated N-terminus of the latter. It can be directly connected or connected through a joint. Regarding the connection point as the origin, the N terminal side of the origin is negative, and the C terminal side is positive.

The following shows the amino acid sequence 453-469 of HPV6 L1 protein and the corresponding sequence of multiple types of HPV L1 protein. It can be seen that these sequences are highly similar. This sequence overlaps with the α5 region. The number in parentheses indicates the position of the last amino acid of the listed sequence. For HPV type 45, some HPV type 45 strains have an additional 26 amino acids at the N-terminus of the L1 protein, while in other HPV type 45 strains There is no such additional 26 amino acids at the N-terminus of the L1 protein, so it is expressed as (478)+26.

HPV6 ELDQYPLGRKFLLQSGY(469)

HPV11 ELDQFPLGRKFLLQSGY(470)

HPV16 DLDQFPLGRKFLLQAGL(474)

HPV18 DLDQYPLGRKFLVQAGL(475)

HPV31 DLDQFPLGRKFLLQAGY(475)

HPV35 DLDQFPLGRKFLLQAGL(472)

HPV39 ELDQFPLGRKFLLQARV(474)

HPV45 DLDQYPLGRKFLVQAGL(478)+26

HPV51 DLDQFALGRKFLLQVGV(474)

HPV52 DLDQFPLGRKFLLQAGL(478)

HPV56 DLDQFPLGRKFLMQLGTRS(474)

HPV58 DLDQFPLGRKFLLQSGL(473)

HPV33 DLDQFPLGRKFLLQAGL(473) KAKPKLKRAAPTSTRTSSAKRKKVKK among them, 480-481 KR and 493-496 KRKK is the nuclear localization sequence.

HPV59 DLDQFPLGRKFLLQLGA(475)RPKPTIGPRKRAAPAPTSTPSPKRVKRR KSSRK, of which RKR 484-486 and KRVKRRK 498-504 are nuclear localization sequences Column.

In one embodiment of the present invention, the inventors conveniently completed the C-terminal replacement of the L1 protein between the different types with the help of the sequence similarity of the α5 region and its surrounding regions between multiple HPV types.

In the most preferred embodiment of the present invention, the inventors noticed that each type of HPV L1 protein has a tetrapeptide RKFL in a similar position, and a more favorable situation is a hexapeptide LGRKFL. The inventor cleverly used this highly conserved sequence to design the connection point of the chimeric protein at any amino acid position of this oligopeptide. From one aspect, the sequence from the N-terminus of the chimeric protein to RKFL or LGRKFL is the same as the sequence of the N-terminal fragment derived from the first type of HPV L1 protein, while on the other hand, it is from RKFL or LGRKFL to the chimeric protein. The C-terminal end of the synthin has the same sequence as the C-terminal fragment derived from the second type of L1 protein.

The chimeric protein thus produced maintains a high degree of similarity with the natural HPV L1 protein, and it can be expected that it will perform well in the production and subsequent medical or preventive processes.

Those skilled in the art will understand that because there are different strains of HPV of the same type, their natural sequences are different, and chimeric proteins constructed from different strains also fall into the present invention.

Those skilled in the art will understand that because of the high similarity of different types of HPV L1, if the chimeric protein is constructed, the N-terminal fragment derived from the first type of HPV L1 protein will extend more amino acids to the C-terminal. Residues, or the C-terminal fragment derived from the HPV L1 protein of the second type extends more amino acid residues to the N-terminal, and it is also possible that the same or similar amino acids at the corresponding positions form the structure of the present invention Consistent chimeric protein. The chimeric protein thus formed also falls into the present invention.

Those skilled in the art will understand that, on the basis of the chimeric protein of the above-described embodiment, variants of the chimeric protein may be formed through mutation, deletion and/or addition of amino acid residues. These variants may have the immunogenicity of the first type of HPV L1 protein, can form VLPs, and have good yield and solubility. The chimeric protein thus formed also falls into the present invention.

The beneficial effects of the invention

The expression systems commonly used for the production of virus-like particles are divided into eukaryotic expression systems and prokaryotic expression systems. The papillomavirus L protein expressed by the eukaryotic expression system can spontaneously assemble into virus-like particles, but it has the disadvantage of low expression and difficult mass production. The natural conformation of the papillomavirus L protein expressed by the prokaryotic expression system is often destroyed, and later in vitro processing is required to obtain virus-like particles, and the yield is low, making it difficult to industrialize.

The present invention transforms the C-terminus of the L protein of papillomavirus (such as human papillomavirus), for example, replacing it with HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1 protein, or HPV 68 The C-terminal fragment in the type L1 protein can increase the expression and solubility of the papillomavirus L protein in an expression system (for example, host cells, such as insect cells). This can be used for large-scale production of vaccines such as HPV vaccines.

The inventor found that HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1 protein, and HPV type 68 L1 protein are better in expression and solubility than other types of L1 protein, and found The increased protein expression and solubility depend on the C-terminal sequence of the HPV L1 protein. In the 107 type HPV L1 protein, most of them have a nuclear localization sequence at the C-terminal, and the C-terminal sequence has a certain similarity.

For the papillomavirus L protein that cannot be expressed at present, the expression level is very low, or the expression is insoluble, replace its C-terminal fragment with HPV type 16 L1 protein, HPV type 28 L1 protein, HPV type 33 L1 protein, HPV type 59 L1 The C-terminal fragment of the protein or HPV type 68 L1 protein makes it possible to soluble expression and subsequent purification of papilloma L protein, which is originally very low or insoluble. This can be used for the large-scale production of more valent vaccines (such as HPV vaccines), making it possible to prevent multiple papillomaviruses, especially HPV infections more comprehensively.

In order to achieve large-scale production of vaccines, there is a need to further increase the expression and solubility of HPV 16 L1 protein in insect cells. In addition, in yeast cells, because the disulfide bonds cannot be formed correctly, the virus-like particles assembled by HPV type 16 L1 protein lack a good conformation.

The present invention transforms the C-terminal fragment of HPV type 16 L1 protein into the C-terminal fragment of HPV type 33 L1 protein. The modified chimeric HPV type 16 L1 protein will be compared with the HPV type 16 L1 protein before the modification in insects. The expression and solubility in the cells are greatly improved. It can be used for large-scale production of HPV 16 vaccine. In addition, the chimeric HPV type 16 L1 protein can correctly form disulfide bonds in insect cells and assemble into HPV type 16 virus-like particles with a good conformation. This can improve the immunogenicity of HPV 16 virus-like particles and produce a better immune response.

definition

Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention belongs. To facilitate the understanding of the present invention, the usual meanings of the following terms are quoted below.

When used herein and in the appended claims, the singular forms "a/kind", "another/kind" and "said/the" include plural references unless the context clearly dictates otherwise. Unless expressly stated otherwise, the terms "include/include/have", "for example", etc. are intended to convey inclusion rather than limitation.

The term "immunogenicity" refers to the ability of a substance, such as a protein or polypeptide, to stimulate an immune response, that is, the ability to stimulate the production of antibodies, especially the production of body fluids or to stimulate a cell-mediated response.

The term "antibody" refers to an immunoglobulin molecule capable of binding an antigen. Antibodies can be polyclonal mixtures or monoclonal. The antibody may be a whole immunoglobulin derived from a natural source or a recombinant source or may be an immunoreactive part of a whole immunoglobulin. Antibodies can exist in a variety of forms, including, for example, Fv, Fab', F(ab')2, and as a single chain.

The term "antigenicity" refers to the ability of a substance, such as a protein or polypeptide, to produce antibodies that specifically bind to it.

The term "epitope" includes any protein determinant capable of specifically binding to an antibody or T cell receptor. Epitope determinants usually consist of chemically active surface groups of molecules (for example, amino acids or sugar side chains, or combinations thereof), and usually have specific three-dimensional structural characteristics and specific charge characteristics.

The terms "subtype" or "type" are used interchangeably herein, and refer to genetic variants of the viral antigen so that a subtype can be recognized by the immune system as being distinguished from a different subtype. For example, HPV 16 can be distinguished from HPV 33 in immunology.

The term "HPV L1 protein" is used herein, and the terms "HPV" and "human papilloma virus" refer to non-enveloped double-stranded DNA viruses of the papillomavirus family. Their genomes are round and are about 8 kilobase pairs in size. Most HPV encode eight major proteins, six are located in the "early" region (E1-E2), and two are located in the "late" region (L1 (major capsid protein) and L2 (minor capsid protein)). More than 120 HPV types have been identified and they are numbered (for example, HPV-16, HPV-18, etc.).

The term "HPV" or "HPV virus" refers to the papillomavirus of the papillomavirus family. It is a non-enveloped DNA virus. The viral genome is a double-stranded closed-loop DNA with a size of about 8kb. It can usually be divided into three regions: ①Early region (E), containing 6 open reading frames encoding E1, E2, E4～E7 virus replication, transcription and transformation related non-structural proteins, and E3 and E8 open reading frames; ②Late region (L) contains codes The reading frame of the major capsid protein L1 and the minor capsid protein L2; ③Long regulatory region (LCR) does not encode any protein, but has the origin of replication and multiple transcription factor binding sites.

The terms "HPV L1 protein" and "HPV L2 protein" refer to proteins encoded by the late region (L) of the HPV gene and synthesized late in the HPV infection cycle. The L1 protein is the main capsid protein and has a molecular weight of 55-60 kDa. L2 protein is the minor capsid protein. 72 L1 pentamers constitute the outer shell of icosahedral HPV virus particles, which enclose the closed-loop double-stranded DNA microchromosomes. The L2 protein is located inside the L1 protein.

The term "virus-like particle" is a hollow particle containing one or more structural proteins of a certain virus without viral nucleic acid.

"HPV pseudovirus" utilizes the characteristic of HPV VLP to wrap nucleic acid non-specifically, and is formed by wrapping free DNA or introducing foreign plasmids into VLP composed of HPV L1 and L2 expressed in cells. It is an ideal HPV neutralization experimental model in vitro.

"Pseudovirus neutralization method" is a method to evaluate the neutralizing activity of antibodies. After immunized animal serum is incubated with a certain amount of pseudovirus, the cells will be infected. The cells will increase with the increase of neutralizing antibodies in the serum. Decrease, there may be a linear negative correlation within a certain range, so the neutralizing activity of antibodies in serum can be evaluated by detecting changes in the number of expressing cells.

The term "fragment thereof" or "variant thereof" means that a part of the nucleotide or amino acid sequence according to the present invention is deleted, inserted and/or substituted. Preferably, the fragments or variants of the polypeptides provided by the present invention can trigger humoral and/or cellular immune responses in animals or humans.

The term "chimeric" means that polypeptides or nucleotide sequences derived from different parent molecules are linked together via amide bonds or 3', 5'-phosphodiester bonds, respectively. Preferably, they are not separated by additional linker sequences, but are directly adjacent to each other.

The term "truncated" means by removing one or more amino acids from the N and/or C-terminus of the polypeptide or deleting one or more amino acids within the polypeptide.

The term "nuclear localization sequence" is an amino acid sequence that can guide a protein into the nucleus. In some HPV L1 proteins, two close basic residue clusters (ie, nuclear localization sequence) (for example, one is KRKR, KRKK, KRKRK, KRKKRK, KRVKRRK, etc., and the other is KR, RKR, KRK, etc.) Spacer of 10-14 amino acids. The above-mentioned basic residue cluster belongs to the nuclear localization sequence. In other HPV L1 proteins, the nuclear localization sequence is a compact cluster of basic residues formed by arginine and/or lysine. Nuclear localization sequences include, but are not limited to, examples of basic residue clusters as described above. See Jun Yang, etc., Predicting the Nuclear Localization Signals of 107 Types of HPV L1 Proteins by Bioinformatic Analysis, Genomics, Proteomics & Bioinformatics Volume 4, Issue 1, 2006, Pages 34-41, all of which are incorporated herein by reference.

The term "functional variant" refers to a version of a polypeptide or protein that has been truncated, mutated, deleted, and/or added and still retains the desired activity or characteristics.

The "sequence identity" between two polypeptide or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the compared molecules. When calculating the percent identity, the sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) . The above program can be publicly obtained from the International Center for Biotechnology Information (NCBI) and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.

Non-critical amino acids can be conservatively substituted without affecting the normal function of the protein. Conservative substitution means replacing an amino acid with a chemically or functionally similar amino acid. It is well known in the art to provide conservative substitution tables for similar amino acids. For example, in some embodiments, the amino acid groups provided in Tables 1-3 are considered to be mutually conservative substitutions.

Table 1 In some embodiments, selected groups of amino acids that are considered to be conservatively substituted for each other

Acid residues	D and E
Basic residues	K, R and H
Hydrophilic uncharged residues	S, T, N and Q
Aliphatic uncharged residues	G, A, V, L and I
Non-polar uncharged residues	C, M and P
Aromatic residues	F, Y and W

Table 2 In certain embodiments, other selected groups of amino acids that are considered to be conservative substitutions for each other

Group 1	A, S and T
Group 2	D and E
Group 3	N and Q
Group 4	R and K
Group 5	I, L and M
Group 6	F, Y and W

Table 3 In certain embodiments, other selected groups of amino acids that are considered to be conservative substitutions for each other

Group A	A and G
Group B	D and E
Group C	N and Q
Group D	R, K and H
Group E	I, L, M, V

Group F	F, Y and W
Group G	S and T
Group H	C and M

The term "amino acid" means twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C ); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine ( Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

The term "adjuvant" refers to a compound or mixture that enhances the immune response. In particular, the vaccine may contain adjuvants. The adjuvant used in the present invention may include, but is not limited to, one or more of the following: mineral adjuvant compositions, oil-milk adjuvants, saponin adjuvant preparations, bacteria or microbial derivatives.

The term "vector" means a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure and as a vector integrated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked by such vectors.

The term "host cell" means a cell into which exogenous nucleic acid has been introduced, and the progeny of such a cell. Host cells include "transformants" (or "transformed cells"), "transfectants" (or "transfected cells"), or "infectants" (or "infected cells"), each of which includes primary transformation, transfection, or Infected cells and their descendants. Such offspring may not be exactly the same as the parent cell in nucleic acid content, and may contain mutations.

The amount of administration is preferably a "prophylactically effective amount" (prevention can be regarded as a treatment herein, and the two are used interchangeably), which is sufficient to show a benefit to the individual.

Example

Example 1: Construction of a chimeric gene where HPV16L1 C-terminal is replaced with HPV33L1 C-terminal

1.1 Construction of pFB-HPV16L1 used as a template

Entrusted Thermo Fisher Company [former Yingwei Jieji (Shanghai) Trading Co., Ltd.] to synthesize the HPV16L1 gene, and the synthesized sequence has KpnI and XbaI restriction sites at both ends, and the sequence is shown in SEQ ID NO: 5. The synthesized gene fragment was ligated with pcDNA3 vector (seller Thermo Fisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV16-L1 containing a nucleotide sequence encoding HPV16L1 1-505 amino acids.

The obtained pcDNA3-HPV16-L1 plasmid was digested with KpnI and XbaI to obtain the HPV16L1 (1-505) gene fragment. Then the fragment was ligated with the pFastBac ^TM 1 vector (seller Thermo Fisher) that was digested with KpnI and XbaI to obtain a bacmid vector containing the HPV16L1 (1-505) gene fragment, named pFB-HPV16L1.

1.2 Construction of pFB-HPV33L1 used as a template

Entrusted Thermo Fisher Company [former Yingwei Jieji (Shanghai) Trading Co., Ltd.] to synthesize the HPV33L1 gene, and the synthetic sequence has KpnI and XbaI restriction sites at both ends, and the gene fragment sequence is shown in SEQ ID NO: 6. The synthesized gene fragment was ligated with pcDNA3 vector (seller Thermo Fisher) through KpnI and XbaI restriction sites to obtain a plasmid pcDNA3-HPV33-L1 containing a nucleotide sequence encoding HPV33L1 and 1-499 amino acids.

The obtained pcDNA3-HPV33-L1 plasmid was digested with KpnI and XbaI to obtain the HPV33L1 (1-499) gene fragment. Then the fragment was ligated with the pFastBacTM1 vector (seller Thermo Fisher) double digested with KpnI and XbaI to obtain a bacmid vector containing the HPV33L1 (1-499) gene fragment, named pFB-HPV33L1.

1.3 Construction of pFB-HPV16L1: 33C

Replace HPV16L1 C-terminal with HPV33L1 C-terminal chimeric gene: Using the successfully constructed recombinant plasmid pFB-HPV16L1 as the gene template, primers F1 and R1 are used to amplify a 1441bp gene fragment. The primer sequence F1 is shown in SEQ ID No: 7. R1 is shown in SEQ ID No: 8.

The gene fragment includes a gene fragment encoding 1-474 amino acids of HPV16L1, 10 bases overlapping with the gene fragment of 474-499 amino acids of HPV33L1, and a KpnI restriction site (GGTAC^C) segment. The amplified sequence is as SEQ ID No: 9 shows:

PCR amplification parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 seconds, annealing at 69°C for 15 seconds, 72°C for 1kb/1min, and 30 cycles; extension at 72°C for 5 minutes; ending at 16°C.

Using the recombinant plasmid pFB-HPV33L1 as a gene template, primers F2 and R2 were used to amplify a gene fragment with a length of 101 bp. The primer sequence F2 is shown in SEQ ID No: 10, and R2 is shown in SEQ ID No: 11.

This gene fragment contains the 26 (474-499) amino acid gene fragment of HPV33L1 C-terminal, the 10bp base overlapping with the 1-474 amino acid C-terminal gene fragment of HPV16L1, and the XbaI (T^CTAGA) restriction site, amplified The sequence is shown in SEQ ID No: 12.

PCR amplification parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 seconds, annealing at 69°C for 15 seconds, 72°C for 1kb/1min, and 30 cycles; extension at 72°C for 5 minutes; ending at 16°C.

PCR splicing sequence:

The splicing primers are F1 and R2 respectively, and the fragments amplified by the above primers (fragments amplified by F1 and R1, and fragments amplified by F2 and R2) are used as templates.

PCR splicing parameters: 94℃ pre-denaturation 5min; 98℃ denaturation 10s, 52℃ annealing 15s, 72℃ 1kb/1min, for 5 cycles; 98℃ denaturation 10s, 68℃ annealing 15s, 72℃ 1kb/1min, for 25 cycles Cycle; extend at 72°C for 5 min; end at 16°C.

Finally, SEQ ID NO: 4 is obtained, which encodes a nucleotide sequence consisting of amino acids 1-474 of HPV16L1 and 26 (474-499) amino acids at the C-terminus of HPV33L1, with KpnI and XbaI restriction sites at both ends (hereinafter referred to as splicing sequence).

The pFastBac ^TM 1 vector and the splicing sequence fragments were digested with KpnI+XbaI, and the splicing sequence was cloned into the pFastBac ^TM 1 vector to obtain the recombinant plasmid pFB-HPV16L1:33C. It is a chimeric gene in which HPV16L1 C-terminal is replaced with HPV33L1 C-terminal.

Example 2: HPV 16L1: 33C recombinant baculovirus packaging

After the recombinant plasmid of pFB-HPV 16L1:33C constructed in Example 1 was identified and sequenced correctly, it was transformed into DH10Bac bacterial competent cells (

The kit, purchased from Thermo Fisher), was cultured and amplified at 37°C, and streaked on a plate. White plaque was selected and amplified. After culturing overnight, the bacterial solution was collected and the recombinant bacmid DNA was extracted by alkaline lysis.

It was transfected into insect cells SF9 with cationic transfection reagent (purchased from Sino Biological) for packaging of recombinant baculovirus seeds. The specific operations are as follows:

a. Take the SF9 cells in the logarithmic growth phase to inoculate dish at a density of 0.6×10 ⁶ cell/dish, and place the dish inoculated with SF9 cells at room temperature for 2 hours to adhere to the wall.

b. Add 20 μL Bacmid DNA of the extracted plasmid to 200 μL Grace's Medium (serum-free, no additives, purchased from Gibico), mix and invert 5 times.

c. 25μL 0.2x TF1 (transfection reagent, purchased from Sino Biological) was added dropwise to 200μL Grace's Meduim and mixed gently.

d. Mix b and c. Incubate at room temperature for 15-45min.

e. When DNA is incubated with cellfectin (purchased from Sino Biological), discard the cell supernatant and add Grace Medium 0.8 mL/dish without serum supplement.

f. Drop the DNA and transfection reagent complexes incubated in d into the dish.

g. Incubate at 27°C for 2hr.

h. Discard the cell culture medium and add 2.5mL/dish complete growth medium (SCD6SF+10%FBS) (SCD6SF purchased from Sino Biological, FBS purchased from Gibico).

i. Incubate at 27°C for 7 days to observe whether there is virus infection.

After transfection, the virus supernatant is collected after the cells have obvious lesions, and the culture is generally 7-11 days. Use a pipette to aseptically collect the virus supernatant, which is the HPV16L1:33C P1 generation virus seed. Use HPV16L1: 33C P1 generation virus to infect SF9 cells at a ratio of 1:50 (V/V), the infection density of SF9 cells is 2×10 ⁶ cells/mL, culture and expansion at 27°C for 3 days, 1000g±200g room temperature centrifugation for 10 minutes , The collected virus supernatant is the P2 generation virus and can be used for infection production.

Example 3: HPV 16L1: 33C expression production

The baculovirus containing the HPV 16L1:33C recombinant gene obtained in Example 2 was used to infect High Five cells, the infection ratio was 1:200 (V/V), and the cell pellet was collected by centrifugation at 1000g±100g at room temperature. Use PBS or MOPS buffer ( (pH 6.0-7.0, salt concentration 100mM-1M) ultrasonically lyse the cell pellet, sonicate at low temperature for 3min, centrifuge at a centrifugal force greater than 10000g for 10 minutes, collect the centrifuged supernatant, and detect by SDS-PAGE electrophoresis. Lane 1: Marker (Marker is seven purified proteins with a molecular weight ranging from 14.4 to 116 kDa, and the manufacturer is Thermo Scientific); Lane 2: Cell lysate; Lane 3: Supernatant collected after centrifugation of the lysate.

The results are shown in Figure 1. The HPV 16L1:33C L1 protein produced by this method has a yield of greater than 100 mg/L and a protein size of about 56KD, which can be used for large-scale production.

Example 4: Purification and preparation of HPV 16L1: 33C virus-like particles

The HPV 16L1: 33C virus-like particle purification method is a two-step chromatography method, namely the HS-MMA method. The supernatant collected in Example 3 is purified to obtain high-purity virus-like particles.

The first step of chromatography:

Medium: using Thermo Fisher

50HS strong cation exchange medium.

Medium volume: medium volume 150mL, linear flow rate 30mL/min.

Chromatography conditions: equilibration buffer (pH 6.2, salt concentration of 50 mM phosphate, 0.5 M sodium chloride); washing buffer (salt concentration of 50 mM phosphate, 0.75 M sodium chloride, pH 6.2;)

The column is first equilibrated with 5CV buffer and then loaded. After loading the sample, 5CV equilibration buffer and washing buffer were used to elute the contaminated proteins.

Elution conditions: pH 6.2, elution salt concentration of 1.25 M sodium chloride, elution with 50 mM phosphate buffer containing 50 mM arginine hydrochloride.

The second step of chromatography:

Medium: MMA ion exchange medium produced by Shanghai Boglong Company.

Medium volume: medium volume 150mL, linear flow rate 30mL/min.

Chromatography conditions: balance buffer 50mM PB, 1.25M NaCl, pH 6.2. The column is first equilibrated with 4CV equilibration buffer and then loaded. After loading the sample, wash the mixed protein with 5CV equilibration buffer, and then use the elution buffer to elute the target protein to collect the protein.

Elution conditions: 100mM NaAC, 150mM NaCl, 0.01% Tween 80, pH 4.5.

Example 5: Morphological detection of HPV 16L1: 33C virus-like particles

Take 10μL sample for transmission electron microscope observation. Fix the sample on a carbon-sprayed copper mesh for 2 minutes, absorb the remaining liquid with filter paper, and stain with phosphotungstic acid (Beijing Zhongjing Science and Technology Co., Ltd., concentration 2%, pH 6.5) twice, each for 30 seconds. The residual staining solution is absorbed with filter paper, and it can be observed under a transmission electron microscope after drying. The transmission electron microscope (brand: Hitachi, model: H-7650) is 80KV, and the magnification is 80,000 times. The results of the electron microscope observation are shown in Figure 2. It can be seen from Figure 2 that the HPV 16L1:33C modified at the C end can form virus-like particles of uniform size, with an average diameter of about 60 nm.

Example 6: Evaluation of animal immunogenicity of HPV 16L1: 33C virus-like particles

6.1 Model establishment of pseudovirus neutralizing cells

Because HPV is difficult to culture in vitro and has strong host specificity, it is difficult to reproduce in organisms other than the human body, and there is a lack of suitable animal models. Therefore, it is necessary to establish a suitable and effective in vitro neutralization experimental model for the evaluation of vaccine immunity.

HPV pseudovirus is an ideal HPV in vitro neutralization experimental model: HPV VLP has the characteristic of non-specifically encapsulating nucleic acid, and the VLP composed of HPV L1 and L2 expressed in cells wraps free DNA or introduces foreign plasmid to form HPV pseudovirus.

The pseudovirus neutralization method was used to analyze the immunogenicity of animal serum samples after immunization. HPV16 virus-like particle samples can produce neutralizing antibodies against HPV16 after immunizing animals, which can neutralize HPV16 pseudoviruses. Incubate the immunized animal serum with a certain amount of pseudovirus and then infect the cells. The cells that can express GFP fluorescence will decrease with the increase of neutralizing antibodies in the serum. There may be a linear negative correlation within a certain range, so The neutralizing activity of antibodies in serum can be evaluated by detecting changes in the number of cells expressing GFP.

Pseudovirus construction method: HPV16 type pCMV3-3-HPV16L1+L2 (L1 sequence comes from Uniprot P03101, L2 sequence comes from Uniprot P03107) plasmid (purchased from Sino Biological) and fluorescent plasmid (PSEU-GFP Spark, purchased from Sino Biological) ) Was co-transfected into 293FT adherent cells (purchased from Thermo Fisher). References for specific methods (Pastrana D V, Buck C B, Pang Y S, Thompson C D, Castle P E, FitzGerald P C, Kjaer S K, Lowy D R, Schiller J T. Reactivity of human sera in a sensitive, high -throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18. [J]Virology 2004,321:205-216.). The pseudovirus supernatant was collected and aliquoted, and stored in a refrigerator at -80°C for later use.

6.2 Evaluation of Animal Immune Protection of HPV 16L1: 33C Virus-like Particles

Mouse immunization program:

HPV 16L1: 33C virus-like particles were adsorbed on aluminum phosphate adjuvant, and 200 μL was used to immunize mice after mixing. The immunization dose of each mouse was 0.15 μg, and 10 mice were immunized on day 0 and day 7 of the experiment. On the 21st day, the diluted samples were used to immunize the mice. At the same time, a blank serum control group was set up. On the 28th day of the experiment, the mice’s eyeballs were taken for blood, and the serum was separated for pseudovirus neutralization titer detection.

Mouse EC50 detection:

After the mouse serum was inactivated at 56°C for 30 minutes, it was centrifuged at 6000 g, and the supernatant was taken for detection after 5 minutes. 4-8 hours before the detection, 293FT cells were plated in a 96-well plate at a density of 15000 cells/well and cultured in a carbon dioxide incubator at 37°C and 5% CO ₂ . After immunization, mouse serum and blank control serum were serially diluted with neutralization medium and mixed with the HPV16 pseudovirus prepared in 6.1 at a volume ratio of 1:1. After incubating in the refrigerator at 2～8℃ for 1 hour, add 100μL/well to the 293FT cells plated 4-8 hours in advance, 2 replicate wells for each sample, and set up a blank serum control well, a false virus positive control well and a negative control at the same time hole. After adding the sample, the cells were cultured in a carbon dioxide incubator at 37°C and 5% CO ₂ for 62-96 hours, and then fluorescence scanning and photographing were performed in an enzyme-linked spot analyzer (model: S6Universal-V Analyzer, manufacturer: CTL). count. Each mouse serum samples by calculating the inhibition rate, calculated according to Reed-Muench method suppresses serum obtained and the maximum serum dilution factor was 50%, i.e., half of the effective dilution EC _50.

The detection results of HPV16 serum pseudovirus neutralization titer are shown in Table 4.

Table 4 EC ₅₀ (GMT±SEM) of mouse serum neutralization titer

Remarks:

1. Number of animals, N=10;

2. GMT (Geometric Mean Titer): geometric mean titer;

3. SEM (Standard Error of Mean): Standard error.

The above test results show that the HPV 16L1:33C virus-like particles prepared by the present invention have good immunogenicity, can produce high-titer neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

Comparative Example 1: Expression of HPV16L1 (1-474) with C-terminal truncation

The inventor tried to shorten the C-terminus of HPV16L1 by 31 amino acids, and named it HPV16L1(1-474) (SEQ ID NO:1). However, it was found in the research that the truncated HPV16L1(1-474) protein has high expression level but poor protein solubility, and it is difficult to extract and purify. The specific expression and extraction results are shown in Figure 3.

Although the foregoing has described the present invention in detail by way of instructions and embodiments, its purpose is to facilitate understanding. It is obvious that those skilled in the art can make various modifications and improvements to the technical solution of the present invention without departing from the additional The spirit or scope of the claims.

Appendix 1: Sequence Listing

Claims (23)

1. A chimeric HPV type 16 L1 protein, from its N-terminus to C-terminus, contains:

   a. N-terminal fragment derived from HPV type 16 L1 protein, said N-terminal fragment maintains the immunogenicity of HPV type 16 L1 protein; and

   b. The C-terminal fragment derived from the L1 protein of the second type papillomavirus, which has the characteristics of better expression and solubility than other types of L1 protein;

   The chimeric HPV type 16 L1 protein has the immunogenicity of HPV type 16 L1 protein.
2. The chimeric HPV type 16 L1 protein according to claim 1, wherein

   The N-terminal fragment is a fragment obtained by truncating the C-terminus of the natural sequence of HPV type 16 L1 protein to any amino acid position in its α5 region, and a fragment having at least 98% identity therewith; and

   The C-terminal fragment is a fragment obtained by truncating the N-terminus of the natural sequence of the second type papillomavirus L1 protein to any amino acid position in its α5 region, and this fragment is further mutated, deleted and/ Or add functional variants.
3. The chimeric HPV type 16 L1 protein of claim 2, wherein the C-terminal fragment contains one or more nuclear localization sequences.
4. The chimeric HPV type 16 L1 protein of claim 1, wherein the second type of papillomavirus L1 protein is selected from HPV type 1, type 2, type 3, type 4, type 6, type 7, 10 Type, Type 11, Type 13, Type 16, Type 18, Type 22, Type 26, Type 28, Type 31, Type 32, Type 33, Type 35, Type 39, Type 42, 44, 45, 51, Type 52, 53, 56, 58, 58, 59, 60, 63, 66, 68, 73 or 82 L1 protein;

   Preferably, the L1 protein of the second type of papillomavirus is selected from HPV type 16, type 28, type 33, type 59, or type 68 L1 protein;

   More preferably, the L1 protein of the second type of papillomavirus is selected from HPV type 33 or HPV type 59 L1 protein.
5. The chimeric HPV type 16 L1 protein according to claim 4, wherein the C-terminal fragment is SEQ ID No: 2; or a fragment of m amino acids in length, preferably covering the first 1 of SEQ ID No: 2. A fragment of the amino acid at position m; where m is an integer from 8 to 26.
6. The chimeric HPV type 16 L1 protein according to claim 4, wherein the C-terminal fragment is SEQ ID No: 13; or a fragment with a length of n amino acids, preferably covering the first 1 of SEQ ID No: 13 A fragment of amino acid at position n; where n is an integer from 16 to 38.
7. The chimeric HPV type 16 L1 protein according to claim 1, wherein the N-terminal fragment is obtained by truncating the C-terminus of the sequence shown in SEQ ID No:1 to any amino acid position in its α5 region The fragments have 98%, 98.5%, 99%, 99.5% or 100% identity.
8. The chimeric HPV type 16 L1 protein according to claim 1, wherein the C-terminus of the N-terminal fragment and the N-terminus of the C-terminal fragment are directly connected or connected through a linker.
9. The chimeric HPV type 16 L1 protein according to claim 1, wherein when the C-terminus of the N-terminal fragment is connected to the N-terminus of the C-terminal fragment, it is located between plus and minus 4 amino acids The following contiguous amino acid sequence exists in the range: RKFL;

   Preferably, the following continuous amino acid sequence exists within the range of plus or minus 6 amino acid positions of the connection point: LGRKFL.
10. The chimeric HPV type 16 L1 protein according to claim 1, which has 98%, 98.5%, 99%, 99.5% or 100% identity with SEQ ID No: 3.
11. An HPV type 16 virus-like particle comprising the chimeric HPV type 16 L1 protein of any one of claims 1 to 10.
12. The HPV type 16 virus-like particle of claim 11, which is an icosahedron composed of 72 pentamers of the chimeric HPV type 16 L1 protein.
13. An immunogenic composition for preventing HPV-related diseases or infections, which comprises the HPV type 16 virus-like particles according to claim 11 or 12 and an adjuvant.
14. An isolated polynucleotide encoding the chimeric HPV type 16 L1 protein of any one of claims 1 to 10.
15. An isolated polynucleotide having the sequence shown in SEQ ID No: 4.
16. A vector comprising the polynucleotide according to claim 14 or 15.
17. The vector according to claim 16, wherein the vector is a baculovirus vector.
18. A baculovirus comprising the polynucleotide according to claim 14 or 15.
19. A host cell comprising the polynucleotide according to claim 14 or 15, the vector according to claim 16 or 17, or the baculovirus according to claim 18.
20. The host cell according to claim 19, which is an insect cell, preferably, the insect cell is selected from Sf9 cell, Sf21 cell, Hi5 cell and S2 cell.
21. A method for preparing the HPV 16 virus-like particle according to claim 11 or 12, which comprises:

    Culturing the host cell of any one of claims 19 to 20 to express the chimeric HPV type 16 L1 protein and assemble it into virus-like particles; and

    Purify the HPV 16 virus-like particles.
22. The method of claim 21, wherein the host cell is a Hi5 cell.
23. The method according to claim 21, wherein the purification adopts cation exchange chromatography, preferably, the cation exchange chromatography is HS-MMA two-step chromatography.

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