CN114127097A

CN114127097A - Chimeric human papilloma virus 56 type L1 protein

Info

Publication number: CN114127097A
Application number: CN202080051685.6A
Authority: CN
Inventors: 谢良志; 罗春霞; 张伟; 索晓燕; 庞琳; 胡萍
Original assignee: Sinocelltech Ltd
Current assignee: Sinocelltech Ltd
Priority date: 2019-07-19
Filing date: 2020-07-17
Publication date: 2022-03-01
Also published as: WO2021013079A1

Abstract

A chimeric human papillomavirus type 56L 1 protein and a polynucleotide for coding it, and the HPV type 56 virus-like particles and their preparing process are disclosed. The chimeric human papillomavirus type 56L 1 protein comprises an N-terminal fragment derived from the HPV type 56L 1 protein, said N-terminal fragment retaining the immunogenicity of the HPV type 56L 1 protein; and a C-terminal fragment derived from a second type papillomavirus L1 protein, said second type papillomavirus L1 protein having the property of better expression and solubility compared to other types of L1 protein; wherein the chimeric HPV type 56L 1 protein has the immunogenicity of an HPV type 56L 1 protein. The chimeric human papilloma virus 56 type L1 protein has high expression level and solubility, and can be used for large-scale production of vaccines.

Description

Chimeric human papilloma virus 56 type L1 protein

Technical Field

The present invention relates to Human Papillomavirus (HPV) L1 protein and polynucleotides encoding the same, and to HPV virus-like particles and methods for their preparation.

Background

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

The L1 and L2 proteins are synthesized in the middle and late stages of the HPV infection cycle. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is a minor capsid protein. The 72L 1 protein pentamers constituted the outer shell of an icosahedral HPV virion (diameter 45-55nm), which enclosed a 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 Papilomavir 16 Chen, X.S., R.L.Garcea, mol.cell.5(3): 557-.

The ORF of the L1 protein is the most conserved gene in the PV genome and can be used to identify novel PV types. A new PV type is identified to be isolated if the complete genome has been cloned and the DNA sequence of the L1ORF differs by more than 10% from the nearest known PV type. Differences are defined as different subtypes at 2% and 10% homology, and differences of 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 late stages of HPV infection, newly synthesized L1 protein in the cytoplasm is transported into the nucleus of terminally differentiated keratin cells, and together with L2 protein, the replicated HPV genomic DNA is packaged to form infectious virus (Nelson, L.M, et al 2002. nucleic acid antigens of high risk HPV16L1 major capsid protein. J. biol. chem.277: 23958-. This suggests that nuclear introduction of the 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-terminus of the HPV L1 protein, a feature of which is the enrichment of basic amino acids (Garcia-Butos, J., et al 1991.Nuclear protein localization. Biochimica et Biophysica Acta 1071: 83-101).

The 15 High Risk (HR) HPV types can cause cervical, anal, penile, vaginal, vulval, and oropharyngeal cancers. Among them, HPV-16 and HPV-18 types are by far the most common causes of cancer, accounting for about 70% of cervical cancer, with the remainder being caused by other HR-HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82). HPV-16 accounts for approximately 95% of HPV-positive oropharyngeal cancers (OPCs). The persistent low risk genotypes HPV-6 and HPV-11 Cause most anogenital and respiratory papillomas, but are rarely associated with Cancer (Human Papilomavir in Cervical Cancer and Oropharyngeal Cancer: One Cause, Two Diseases Tara. Bernnand John T. Schiller, PhD2 Cancer 2017; 123: 2219-29).

Recombinant expression of the L1 protein using vaccinia virus, baculovirus or yeast systems, the L1 protein self-assembles into virus-like particles (VLPs) containing approximately 72L 1 proteins, similar to the virion coat. VLPs have no indications. VLPs can induce neutralizing antibodies in vaccinated animals, protecting experimental animals against subsequent challenge with infectious virus. Thus, VLPs appear to be excellent candidates for Papillomavirus vaccines (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papilomoavirus 16 Chen, X.S., R.L.Garcea, mol.cell.5(3): 557-.

Of Kulansu Co

Is a bivalent recombinant HPV vaccine. The recombinant baculovirus containing HPV16 type recombinant L1 protein and HPV18 type recombinant L1 protein are obtained through expressing recombinant baculovirus expression vector system in noctuid (Trichoplusia ni) insect cells. The L1 protein self-assembles into virus-like particles for use in the prevention of cervical cancer, grade 2 or 3 cervical intraepithelial neoplasia and carcinoma in situ in 9-25 year old women caused by HPV types 16 and 18, and grade 1 cervical intraepithelial neoplasia (carcinogenic) (https:// www.fda.gov/downloads/biologics bloodvaccins/vaccins/ApplivedProducts/UCM 186981. pdf).

Is a tetravalent (6, 11, 16 and 18 types) recombinant vaccine of human papilloma virus produced by merck company, is used for girls and women of 9 to 26 years old for preventing cervical cancer genital warts (condyloma acuminatum) and precancerous or dysplastic lesions caused by HPV6, 11, 16 and 18 types; and 9-26 year old boys and men for preventing anal cancer, genital warts (condyloma acuminatum) and precancerous or developmental diseases caused by HPV6, 11, 16, 18 typesAbnormal lesions (https:// www.fda.gov/vaccines-blood-biologics/vaccinines/gardasil).

Is a human papilloma virus nine valent recombinant vaccine produced by merck, comprising virus-like particles of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58L 1 proteins, the L1 protein being produced by fermentation of saccharomyces cerevisiae, self-assembled into VLPs. For girls and women aged 9-45 to prevent cervical, vulvar, vaginal and anal cancers caused by HPV16, 18, 31, 33, 45, 52 and 58 types, genital warts (condyloma acuminatum) caused by HPV6 and 11 and precancerous or dysplastic lesions caused by HPV6, 11, 16, 18, 31, 33, 45, 52 and 58 types; and 9-45 year old boys and men for the prevention of anal cancer caused by types 16, 18, 31, 33, 45, 52 and 58, genital warts (condyloma acuminatum) caused by HPV6 and 11, and precancerous or dysplastic lesions caused by HPV6, 11, 16, 18, 31, 33, 45, 52 and 58 (https:// www.fda.gov/vaccines-blood-biologics/vaccines/gardasil-9).

The specification states that HPV types 16 and 18 are responsible for about 70% of cervical cancer, and the remaining 20% of cases are responsible for types 31, 33, 45, 52 and 58

Can prevent 90% of cervical cancer (https:// www.fda.gov/biologics blood lipids/ApplivedPro products/ucm426445. htm).

A key factor in HPV vaccine development is that virus-like particles can be produced in large quantities. The current systems for producing virus-like particles are mainly classified into eukaryotic expression systems and prokaryotic expression systems.

Examples of eukaryotic expression systems that are commonly used include poxvirus expression systems, insect baculovirus expression systems, and yeast expression systems. The natural conformation of the HPV L1 protein expressed in eukaryotic expression systems is less disrupted and can spontaneously assemble to form virus-like particles, but the yield is lower. The prokaryotic expression system mainly comprises an escherichia coli expression system, has high yield, mostly exists in an inclusion body form, is not beneficial to purification, and has a complex production process.

Thus, there remains a need in the art to obtain high yields of HPV virus-like particles.

Disclosure of Invention

In one aspect, the present invention provides a chimeric Human Papillomavirus (HPV) type 56L1 protein comprising, in the N-terminal to C-terminal direction, an N-terminal fragment derived from an HPV type 56L1 protein, said N-terminal fragment retaining the immunogenicity of the HPV type 56L1 protein; a C-terminal fragment derived from a second type of papillomavirus L1 protein, said second type of papillomavirus L1 protein having properties of better expression and solubility compared to other types of L1 protein; wherein the chimeric HPV type 56L1 protein has the immunogenicity of an HPV type 56L1 protein.

In another aspect, the invention provides a HPV type 56 virus-like particle comprising a chimeric HPV type 56L1 protein.

In another aspect, the invention provides an immunogenic composition for the prevention of an HPV-related disease or infection, comprising an HPV type 56 virus-like particle and an adjuvant.

In another aspect, the invention provides an isolated polynucleotide encoding a chimeric HPV type 56L1 protein.

In another aspect, the present invention provides a vector comprising a polynucleotide encoding a chimeric HPV type 56L1 protein.

In another aspect, the present invention provides a baculovirus comprising a polynucleotide encoding a chimeric HPV type 56L1 protein.

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

In another aspect, the present invention provides a method of making a HPV type 56 virus-like particle, comprising culturing a host cell as described above to express the chimeric HPV type 56L1 protein and assemble into a virus-like particle; and purifying the HPV type 56 virus-like particle.

Drawings

FIG. 1 HPV56L 1: expression of 33C L1 protein. M: marker; l: a cell lysate; E-S: supernatant collected after centrifugation of the lysate.

FIG. 2 Transmission Electron microscopy of HPV56L 1:33C virus like particle.

FIG. 3 expression of C-terminally truncated HPV16L1 (1-474). M: marker; l: a cell lysate; E-S: supernatant collected after centrifugation of the lysate.

Detailed Description

In one aspect, the present invention provides a chimeric Human Papillomavirus (HPV) type 56L1 protein comprising, in the N-terminal to C-terminal direction: a. an N-terminal fragment derived from an HPV type 56L1 protein, said N-terminal fragment retaining the immunogenicity of the HPV type 56L1 protein; a C-terminal fragment derived from a second type of papillomavirus L1 protein, said second type of papillomavirus L1 protein having properties of better expression and solubility compared to other types of L1 protein; wherein the chimeric HPV type 56L1 protein has the immunogenicity of an HPV type 56L1 protein.

In one embodiment, the N-terminal fragment is a fragment obtained by truncating the C-terminal end of the natural sequence of the HPV type 56L1 protein shorter than any amino acid position within its α 5 region, and fragments having at least 98% identity thereto; the C-terminal fragment is a fragment obtained by cutting off the N-terminal of the natural sequence of the second type papilloma virus L1 protein to be shorter than any amino acid position in the alpha 5 region thereof, and a functional variant generated by further mutating, deleting and/or adding the fragment.

In another embodiment, the N-terminal fragment has at least 98.5%, 99%, 99.5% or 100% identity to a fragment obtained by truncation of the C-terminus of the native sequence of HPV type 56L1 protein to any one of the amino acid positions within its α 5 region.

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

In one embodiment, the second type papillomavirus L1 protein is selected from HPV type 1, type 2, type 3, type 4, type 6, type 7, type 10, 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, type 44, type 45, type 51, type 52, type 53, type 56, type 58, type 59, type 60, type 63, type 66, type 68, type 73, or type 82L 1 protein;

preferably, the second type papillomavirus L1 protein is selected from HPV type 16, type 28, type 33, type 59, or type 68L 1 protein;

more preferably, said second type papillomavirus L1 protein is selected from the HPV type 33 or HPV type 59L 1 proteins.

In one embodiment, the second type papillomavirus L1 protein is HPV type 33L1 protein and the C-terminal fragment is SEQ ID No. 2; or a fragment thereof of length m amino acids, preferably a fragment encompassing amino acids 1-m of SEQ ID No. 2; wherein m is an integer of 13 to 31.

In one embodiment, the C-terminal fragment of HPV type 33L1 protein has one nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV type 33L1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV type 56L1 protein comprises one or more C-terminal fragments of HPV type 33L1 protein. The C-terminal fragments of the multiple HPV33 type L1 proteins may be the same or different. In one embodiment, the amino acid sequence of amino acid Nos. 7-8 (KR) and the amino acid sequence of amino acid Nos. 20-23 (KRKK) of SEQ ID No. 2 are nuclear localization sequences of the C-terminal fragment of the HPV type 33L1 protein.

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

In one embodiment, the C-terminal fragment of HPV type 59L 1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV type 59L 1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV type 56L1 protein comprises one or more C-terminal fragments of HPV type 59L 1 protein. The C-terminal fragments of the multiple HPV59 type L1 proteins may be the same or different. In one embodiment, the amino acid sequence of amino acid Nos. 14-16 (RKR) and the amino acid sequence of amino acid Nos. 28-34 (KRVKRRK) of SEQ ID No. 13 are nuclear localization sequences of the C-terminal fragment of HPV type 59L 1 protein.

In one embodiment, the chimeric HPV type 56L1 protein comprises both a C-terminal fragment of an HPV type 33L1 protein and a C-terminal fragment of an HPV type 59L 1 protein.

In one embodiment, the N-terminal fragment has 98%, 98.5%, 99%, 99.5% or 100% identity to a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID No.1 to any amino acid position within its α 5 region.

In one embodiment, the C-terminus of the N-terminal fragment is directly linked to the N-terminus of the C-terminal fragment or linked via a linker.

The linker does not affect the immunogenicity of the N-terminal fragment and does not affect the amount of protein expressed or solubility. In one embodiment, the N-terminal fragment and the C-terminal fragment are linked 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 56L1 protein. In another embodiment, the linker may be a partial sequence of HPV type 33L1 protein. In another embodiment, the linker may be a partial sequence of HPV type 59L 1 protein.

In one embodiment, when the C-terminus of the N-terminal fragment is linked to the N-terminus of the C-terminal fragment, the following contiguous amino acid sequence is present within plus or minus 4 amino acid positions of the point of attachment: RKFL; preferably, the following contiguous amino acid sequence is present within plus or minus 6 amino acid positions of the point of attachment: LGRKFL.

In one embodiment, the chimeric HPV type 56L1 protein is 98%, 98.5%, 99%, 99.5% or 100% identical to SEQ ID No. 3.

In another aspect, the invention provides a HPV type 56 virus-like particle comprising a chimeric HPV type 56L1 protein as described above. In one embodiment, the HPV type 56 virus-like particle is an icosahedron composed of a pentamer of 72 of said chimeric HPV type 56L1 proteins. In one embodiment, the HPV type 56 virus-like particle has correctly formed disulfide bonds and thus has a good native conformation. In one embodiment, the HPV type 56 virus-like particle self-assembles in an in vivo expression system.

In one aspect, the invention provides an immunogenic composition for the prevention of an HPV-related disease or infection, comprising a HPV type 56 virus-like particle according to the preceding description and an adjuvant. The prevention may be considered treatment, and the two may be used interchangeably.

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

In one aspect, the invention provides an isolated polynucleotide encoding a chimeric HPV type 56L1 protein as described above. In one embodiment, the polynucleotide is codon optimized for a different expression system. In one embodiment, the polynucleotide is codon optimized for an insect baculovirus expression system.

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

In one aspect, the invention provides a vector comprising a polynucleotide as described previously. In one embodiment, the vector is a baculovirus vector. In one embodiment, the vector may be a transfer vector for a baculovirus expression system. In another embodiment, the vector may be an expression vector for a baculovirus expression system. In another embodiment, the vector may be a recombinant vector for use in a baculovirus expression system.

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

In one aspect, the invention provides a host cell comprising a polynucleotide, vector, or baculovirus as described previously. In one embodiment, the host cell is an insect cell, preferably, the insect cell is selected from the group consisting of Sf9 cell, Sf21 cell, Hi5 cell and S2 cell.

In one aspect, the invention provides a method of preparing a virus-like particle of HPV type 56 according to the foregoing description, comprising: culturing a host cell as described above to express the chimeric HPV type 56L1 protein and assemble into a virus-like particle; and purifying the HPV type 56 virus-like particle.

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 56L1 protein self-assembles into HPV type 56 virus-like particles in a host cell. In one embodiment, the chimeric HPV type 56L1 protein self-assembles into HPV type 56 virus-like particles in a host cell having an icosahedron composed of a pentamer of 72 of the chimeric HPV type 56L1 protein. In one embodiment, the HPV type 56 virus-like particle has correctly formed disulfide bonds and thus has a good native conformation.

In one embodiment, the purification is by cation exchange chromatography. In one embodiment, the purification is performed using strong cation exchange chromatography. In another embodiment, the purification is performed using weak cation exchange chromatography. In one embodiment, the purification employs a combination of multiple cation exchange chromatography. In one embodiment, the purification is performed using HS strong cation exchange chromatography. In another embodiment, purification is by MMA ion exchange chromatography. In another embodiment, the purification is performed by HS-MMA two-step chromatography.

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

The sequence of the L1 protein for each HPV type is conveniently obtained from https:// www.uniprot.org. Each type of HPV L1 may be derived from different strains, and thus the amino acid sequence has multiple versions, any one of which may be used in the present invention, and in the design and design process of the present invention, the HPV L1 protein sequence of a given type may be different from the sequence used in the examples, but such differences do not affect the judgment and conclusion of the inventors.

It is widely believed by those skilled in the art that the C-terminus of the L1 protein does not contain a major neutralizing epitope, and therefore attempts to increase expression levels by truncating the C-terminus of the HPV L1 protein, for example in U.S. Pat. No. 3, 6361778, 1 to Kurarin corporation, the C-terminus of the HPV16L1 protein is truncated by 1-34 amino acids, preferably 26 amino acids, suggesting that the production of VLPs is increased many-fold, preferably at least 10-fold, and more preferably about 10 to 100-fold. Inspired by this, the inventors tried to truncate the C-terminus of HPV type 16L1 by 31 amino acids, named HPV16L1 (1-474). However, the protein expression amount is high, but the protein solubility is poor, and the extraction and purification are difficult (see a comparative example).

The poor solubility of the protein due to such truncation may be the result of a deletion of the nuclear localization sequence at the C-terminus, and the present invention is not limited to this speculation. The inventor finds that the HPV16 type L1 protein, the HPV 28 type L1 protein, the HPV33 type L1 protein, the HPV59 type L1 protein and the HPV 68 type L1 protein are better in expression amount and solubility compared with other types of L1 proteins in research and production processes, and inspires that the inventor replaces the C end of the HPV type which is not easy to extract or has low expression amount with the C end of the L1 protein which has better expression amount and solubility. Namely, the inventors constructed a chimeric protein: the polypeptide comprises an N-terminal fragment derived from a first type papillomavirus L1 protein (such as HPV L1 protein) and a C-terminal fragment derived from a second type papillomavirus L1 protein (such as HPV L1 protein) from the N-terminal direction to the C-terminal direction, wherein the N-terminal fragment provides immunogenicity of the first type papillomavirus (such as HPV), and the C-terminal fragment provides better expression and solubility characteristics. The two can be directly connected or connected through a joint.

To preserve the immunogenicity of the HPV L1 protein of the first type and to ensure that it is able to form VLPs, the inventors determined the length of the N-terminal fragment of the appropriate HPV L1 protein. The following reports relate to epitope studies of common HPV subtypes:

sunanda Baidiya et al reported that epitopes 48EEYDLQFIFQLCKITLTA65,45RHGEEYDLQFIFQLCKITLTA65,63 LPDPPNKF 69,79PETQRLVWAC88,36PVPGQYDA43,77YNPETQRLVWAC88,188DTGYGAMD195,36PVPGQYDATK45,45 KQDIPPKVSAYQYRVFRVFRV 61,130RDNVSVDYKQTQLCI144and 49YSRHEEY DLQFIF62 of the L1 protein could be used as a tool for designing HPV16 and 18 type vaccines (see Epitope design of L1 protein for vaccine production against Human Papilloma viruses 16 and 18, Bioformation 13(3):86-93 Mar2017, incorporated herein by reference in its entirety).

Katharina Slupetzky et al reported that the regions near aa 282-355 and 351-355 of HPV-16 contributed to the neutralizing epitope, and that the latter was the immunodominant site (see, Chimeric plasmid expression a for infection epitope on capsule surfaces, Journal of General Virology (2001),82, 2799-2804, incorporated herein by reference in its entirety).

Brooke Bishop et al prepared the following 3 variants of HPV11, 16, 18 and 35L1 proteins: the N-terminal 9 amino acid deletion, the alpha 4 (corresponding to the amino acid residue at position 404-436 of HPV 16) deletion and the C-terminal 31 amino acid deletion of the peptide fragment were reported, the former two could not assemble into VLP, but the latter was not reported to have the phenomenon

(Crystal Structures OF Four Types OF Human Papilomavir L1 Capsule Proteins UNDERSTRANDING THE SPECIFICITY OF NEUTRAZING MONOCLONAL ANTIBODIES, The Journal OF Biological Chemistry, 282, 31803-. The respective Loop regions of the α -helix and β -sheet of the HPV L1 protein of each type can be readily determined by sequence analysis software commonly used in the art. Wherein the alpha helical region comprises an alpha 1 region, an alpha 2 region, an alpha 3 region, an alpha 4 region and an alpha 5 region.

The inventors performed sequence alignments OF HPV L1 Proteins OF 14 Types (type 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and then performed secondary structure predictions according to The literature cited above (Crystal Structures OF Four Types OF Human Papilomavir L1 Capsid Proteins UNDERSTRANDING THE SPECIFICITY OF NEUTRALIZING MONOCLONAL ANTIBODIES, The Journal OF Biological Chemistry, 282,31803 and 31811) with The following results, where The part between The downward arrows corresponds to The region involved in The literature and deleted for The preparation OF variants.

In addition to the methods of sequence alignment used by the inventors, 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 derived from HPV L1 protein of the first type in the following manner: the native sequence of the L1 protein is truncated in its α 5 region and its vicinity, leaving the sequence from its N-terminus to the newly generated C-terminus of the α 5 region. The sequence thus truncated may be rendered immunogenic for this class and is capable of forming VLPs.

The N-terminal fragment derived from HPV L1 protein of the first type may be further engineered to ensure its immunogenicity and limited ability to form VLPs.

The inventors determined the length of the C-terminal fragment derived from HPV L1 protein of the second type in the following manner. The native sequence of the L1 protein is truncated in its α 5 region and its vicinity, and the newly generated N-terminal to C-terminal sequence from its α 5 region is retained. Such truncated sequences do not have a major neutralizing epitope and do not interfere with the immunogenicity of the resulting chimeric protein.

The C-terminal fragment derived from HPV L1 protein of the second type may further be mutated, deleted and/or added, preferably retaining at least one of its nuclear localization sequences. Yang et al predicted the nuclear localization sequences of 107 HPV subtypes (Yang et al, predicting the nuclear localization signals of 107 types of HPV L1 proteins by biochemical analysis. Geno. prot. bioinfo. Vol.4 No. 12006 is incorporated herein by reference in its entirety), and the nuclear localization sequences of HPV L1 proteins of each type can be readily determined by sequence analysis software commonly used in the art.

The ligation of the N-terminal fragment and the C-terminal fragment occurs at the former newly generated C-terminus and the latter newly generated N-terminus. Either directly or through a linker. Regarding the connection point as the origin, the N-side of the origin is negative, and the C-side thereof is positive.

The amino acid sequences at positions 453-469 of the HPV6L1 protein and the corresponding sequences of the multiple types of HPV L1 protein are shown below. It can be seen that these sequences are highly similar. This sequence coincides with the α 5 region. The numbers in parentheses indicate the position of the last amino acid of the listed sequences, where for HPV type 45, an additional 26 amino acids are present at the N-terminus of the L1 protein of some HPV type 45 strains, while the additional 26 amino acids are absent at the N-terminus of the L1 protein of other HPV type 45 strains, and so are indicated 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 wherein, KR and 493-496 of 480-481 KRKK at position is the nuclear localization sequence.

HPV59

DLDQFPLGRKFLLQLGA(475)RPKPTIGPRKRAAPAPTSTPSPKRVKRR KSSRK, wherein RKR of 484-486 and KRVKRK of 498-504 are nuclear localization sequences And (4) columns.

In one embodiment of the invention, the inventors conveniently accomplished a C-terminal substitution of the L1 protein between different HPV types by virtue of sequence similarity of the α 5 region and its vicinity between the various HPV types.

In the most preferred embodiment of the invention, the inventors note that each HPV type L1 protein has a stretch of tetrapeptide RKFL at a similar position, more advantageously a stretch of hexapeptide LGRKFL. The inventors skillfully utilized this highly conserved sequence to design the point of attachment of the chimeric protein at any amino acid position of this oligopeptide. From one aspect, from the N-terminus of the chimeric protein to RKFL or LGRKFL ending with the N-terminal fragment derived from HPV L1 protein of the first type is identical, while from another aspect from RKFL or LGRKFL to the C-terminus of the chimeric protein is identical to the sequence derived from the C-terminal fragment of L1 protein of the second type.

The chimeric proteins so produced retain a high degree of similarity to the native HPV L1 protein and are expected to perform well during manufacture and even later in the medical or prophylactic process.

It will be understood by those skilled in the art that since the same type of HPV has different strains and thus different natural sequences, chimeric proteins constructed using different strains also fall within the present invention.

It will be appreciated by those skilled in the art that because of the high similarity of the different types of HPV L1, if during the construction of the chimeric protein the N-terminal fragment derived from the first type of HPV L1 protein is extended more amino acid residues C-terminally or the C-terminal fragment derived from the second type of HPV L1 protein is extended more amino acid residues N-terminally, it is also possible to form a chimeric protein structurally identical to the present invention due to the identity or similarity of the amino acids at the corresponding positions. Chimeric proteins so formed are also within the scope of the invention.

Those skilled in the art will appreciate that, on the basis of the chimeric proteins of the above-described embodiments, variants of the chimeric proteins will be formed by mutation, deletion and/or addition of amino acid residues. These variants are likely to have the immunogenicity of the HPV L1 protein of the first type, can form VLPs, and have good yield and solubility. Chimeric proteins so formed are also within the scope of the invention.

Advantageous effects of the invention

Expression systems currently in common use for the production of virus-like particles are divided into eukaryotic and prokaryotic expression systems. The papillomavirus L protein expressed by a eukaryotic expression system can spontaneously assemble into virus-like particles, but has the defect of low expression level and difficult scale production. The natural conformation of the papillomavirus L protein expressed by a prokaryotic expression system is often destroyed, and the virus-like particles can be obtained only by in vitro treatment at a later stage, and the yield is low, so that the industrialization is difficult to realize.

The invention modifies the C end of the L protein of papilloma virus (such as human papilloma virus), for example, the C end fragment in HPV16 type L1 protein, HPV 28 type L1 protein, HPV33 type L1 protein, HPV59 type L1 protein or HPV 68 type L1 protein is replaced, and the expression quantity and the solubility of the L protein of papilloma virus can be improved in an expression system (such as host cells, for example insect cells). This is useful for large scale production of vaccines such as HPV vaccines.

The inventor finds out that the HPV type 16L1 protein, the HPV type 28L 1 protein, the HPV type 33L1 protein, the HPV type 59L 1 protein and the HPV type 68L 1 protein have better expression quantity and solubility compared with the L1 protein of other types, and finds out that the increased protein expression quantity and solubility are dependent on the C-terminal sequence of the HPV L1 protein. Among the HPV type 107L 1 proteins, most have a nuclear localization sequence at the C-terminus, and the C-terminal sequences have some similarity.

For the papillomavirus L protein which cannot be expressed at present, has very low expression level or is insoluble after expression, the C-terminal fragment of the papillomavirus L protein is replaced by the HPV16 type L1 protein, the HPV 28 type L1 protein, the HPV33 type L1 protein, the HPV59 type L1 protein or the C-terminal fragment of the HPV 68 type L1 protein, so that the soluble expression and the subsequent purification of the papillomavirus L protein with very low expression level or insoluble can be possible. This can be used for large scale production of more valuable vaccines (e.g. HPV vaccines), making it possible to more fully prevent infection by a variety of papillomaviruses, particularly HPV.

The HPV56 type L1 protein has low expression level and poor solubility in insect cells, and is not beneficial to subsequent purification and vaccine production. Furthermore, in yeast cells, the virus-like particles assembled from HPV type 56L1 protein lack good conformation because disulfide bonds are not formed correctly.

Compared with the HPV56 type L1 protein before modification, the expression quantity and the solubility of the chimeric HPV56 type L1 protein in insect cells are greatly improved. Can be used for large-scale production of HPV56 type vaccine. In addition, the chimeric HPV type 56L1 protein can correctly form disulfide bonds in insect cells to assemble into HPV type 56 virus-like particles with good conformation. This may increase the immunogenicity of the HPV type 56 virus-like particle, resulting in a better immune response.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the convenience of understanding the present invention, the following terms are referred to in their ordinary meanings.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "comprising/including/having," "for example," and the like are intended to convey inclusion without limitation, unless expressly stated otherwise.

The term "immunogenic" refers to the ability of a substance, such as a protein or polypeptide, to stimulate an immune response, i.e., the ability to stimulate the production of antibodies, particularly the production of a humoral or cell-mediated response.

The term "antibody" refers to an immunoglobulin molecule that binds an antigen. The antibody may be a polyclonal cocktail or a monoclonal. The antibody may be an intact immunoglobulin derived from a natural source or from a recombinant source or may be an immunoreactive portion of an intact immunoglobulin. Antibodies can exist in a variety of forms, including, for example, Fv, Fab ', F (ab') 2, and as single chains.

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

The term "epitope" includes any protein determinant capable of specific binding to an antibody or T cell receptor. Epitopic determinants are typically composed of chemically active surface groups of molecules (e.g., amino acids or sugar side chains, or combinations thereof) and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

The terms "subtype" or "type" are used interchangeably herein to mean a genetic variant of the viral antigen such that one subtype is recognized by the immune system differently from a different subtype. For example, HPV16 is immunologically distinct from HPV 33.

The term "HPV L1 protein" as used herein, the terms "HPV" and "human papilloma virus" refer to non-enveloped double-stranded DNA viruses of the papilloma virus family. Their genome is circular and is about 8 kilobase pairs in size. Most HPVs encode eight major proteins, six located in the "early" region (E1-E2), and two located in the "late" region (L1 (major capsid protein) and L2 (minor capsid protein)). Over 120 HPV types have been identified and are indicated by numbers (e.g., HPV-16, HPV-18, etc.).

The term "HPV" or "HPV virus" refers to papillomaviruses of the papillomavirus family, non-enveloped DNA viruses whose genome is a double-stranded closed-loop DNA of approximately 8kb in size, and can be generally divided into three regions: early region (E) containing 6 open reading frames encoding nonstructural proteins involved in replication, transcription and transformation of viruses E1, E2, E4-E7, and E3 and E8 open reading frames; (ii) the late region (L) contains the reading frames encoding the major capsid protein L1 and the minor capsid protein L2; long regulatory region (LCR) does not encode any protein, but has an 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, which are synthesized late in the HPV infection cycle. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is the minor capsid protein. 72L 1 pentamers constituted the outer shell of the icosahedral HPV virion, encapsulating the closed-loop double-stranded DNA minichromosome. 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.

The HPV pseudovirus is formed by wrapping free DNA or introducing exogenous plasmid through VLP composed of HPV L1 and L2 expressed in cells by using the characteristic of non-specific wrapped nucleic acid of HPV VLP. Is an ideal HPV in vitro neutralization experimental model.

The 'pseudovirus neutralization method' is a method for evaluating the neutralizing activity of antibodies, and after immune animal serum is incubated with a certain amount of pseudovirus and then infects cells, the cells can be reduced along with the increase of neutralizing antibodies in the serum, and can be in linear negative correlation within a certain range, so that the neutralizing activity of the antibodies in the serum can be evaluated by detecting the change of the number of expressed 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 invention are capable of eliciting a humoral and/or cellular immune response in an animal or human.

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

The term "truncation" means by removal of one or more amino acids from the N-and/or C-terminus of a polypeptide or deletion of one or more amino acids within a polypeptide.

The term "nuclear localization sequence" is an amino acid sequence that directs a protein into the nucleus of a cell. In some HPV L1 proteins, a spacer of 10-14 amino acids is between two tight clusters of basic residues (i.e., nuclear localization sequences) (e.g., one is krkrkrkrkrkrkr, KRKK, krkrkrkrkrkrk, KRKKRK, krvkrkrrk, etc., and the other is KR, RKR, KRK, etc.). The basic residue cluster described above 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 et al, predictioning the nucleic Localization Signals of 107 Types of HPV L1 Proteins by biological Analysis, Genomics, Proteomics & Bioinformatics Volume 4, Issue 1,2006, Pages 34-41, the entire contents of which are incorporated herein by reference.

The term "functional variant" is a version of a polypeptide or protein that retains a desired activity or characteristic after truncation, mutation, deletion, and/or addition.

"sequence identity" between two polypeptide or nucleic acid sequences means the number of residues that are identical 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. In calculating percent identity, the sequences being compared are aligned in such a way as to produce the largest match between the sequences, and gaps in the alignment (if any) are resolved by a particular algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al, 1990, J.Mol.biol.215: 403-. The above procedures are publicly available 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 substitutions are intended to refer to the replacement of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. For example, in some embodiments, the sets of amino acids provided in tables 1-3 are considered conservative substitutions for one another.

Table 1 in certain embodiments, selected groups of amino acids that are considered conservative substitutions for one another

Acidic residue	D and E
Basic residue	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 residue	F. Y and W

Table 2 in certain embodiments, other selected groups of amino acids that are considered conservative substitutions for one another

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 conservative substitutions for one another

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 the 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 an immune response. In particular, the vaccine may comprise an adjuvant. Adjuvants for use in the present invention may include, but are not limited to, one or more of the following: mineral adjuvant-containing composition, oil-milk adjuvant, saponin adjuvant preparation, and bacterial or microbial derivative.

The term "vector" means a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are 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 to which such vectors are operably linked.

The term "host cell" means a cell into which an exogenous nucleic acid has been introduced, as well as the progeny of such a cell. Host cells include "transformants" (or "transformed cells"), "transfectants" (or "transfected cells") or "infectors" (or "infected cells"), each of which includes primary transformed, transfected or infected cells and progeny derived therefrom. Such progeny may not be identical in nucleic acid content to the parent cell, and may contain mutations.

The amount administered is preferably a "prophylactically effective amount" (where prophylaxis may be considered treatment, the two are used interchangeably) sufficient to show benefit to the individual.

Examples

Example 1: construction of chimeric Gene having HPV56L 1C-terminal replaced with HPV33L 1C-terminal

1.1 construction of pFB-HPV56L1 used as template

The HPV56L1 gene was synthesized from a gene of Thermo Fisher (Elite Weiji (Shanghai) trade Co., Ltd.), and the synthesized sequence had KpnI and XbaI cleavage sites at both ends, respectively, and the sequence was shown in SEQ ID NO: 5. The synthesized gene fragment was ligated with pcDNA3 vector (sold by Thermo Fisher) through KpnI and XbaI enzymatic cleavage sites to obtain plasmid pcDNA3-HPV56-L1 containing a nucleotide sequence encoding HPV56L 11-499 amino acids.

The resulting pcDNA3-HPV56-L1 plasmid was cleaved with Kpn I and XbaI to obtain a fragment of the gene of HPV56L1 (1-499). Then the fragment is subjected to double enzyme digestion with KpnI and XbaI to obtain pFastBac^TM1 vector (vendor Thermo Fisher) to obtain bacmid vector containing HPV56L1(1-499) gene fragment, which is named pFB-HPV56L 1.

1.2 construction of pFB-HPV33L1 used as template

The HPV33L1 gene was synthesized from a gene of Thermo Fisher (Elite Weiji (Shanghai) trade Co., Ltd.), and the synthetic sequence had KpnI and XbaI cleavage sites at both ends, respectively, and the sequence of the gene fragment was shown in SEQ ID NO: 6. The synthesized gene fragment was ligated with pcDNA3 vector (sold by Thermo Fisher) through KpnI and XbaI enzymatic cleavage sites to obtain plasmid pcDNA3-HPV33-L1 containing the nucleotide sequence encoding amino acids L11-499 of HPV 33.

The resulting pcDNA3-HPV33-L1 plasmid was cleaved with KpnI and XbaI to obtain a fragment of the gene of HPV33L1 (1-499). Then the fragment was ligated with a KpnI and XbaI double digested pFastBacTM1 vector (sold by Thermo Fisher) to obtain a bacmid vector containing HPV33L1(1-499) gene fragment, which was named pFB-HPV33L 1.

1.3 pFB-HPV56L 1: construction of 33C

HPV56L 1C terminal was replaced with HPV33L 1C terminal chimeric gene: a successfully constructed recombinant plasmid pFB-HPV56L1 is used as a gene template, a gene fragment with the length of 1420bp is amplified by primers F1 and R1, the sequence of the primer F1 is shown as SEQ ID No. 7, and the sequence of the primer R1 is shown as SEQ ID No. 8.

The gene fragment comprises a gene fragment which codes 1-467 amino acids of HPV56L1, 10 bases which are overlapped with a gene fragment of 469-499 amino acids of HPV33L1 and a KpnI enzyme cutting site (GGTAC ^ C), and the amplified sequence is shown as SEQ ID No. 9:

PCR amplification parameters: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 69 ℃ for 15s, annealing at 72 ℃ for 1kb/1min, and performing 30 cycles; extending for 5min at 72 ℃; and finishing at 16 ℃.

A gene fragment with the length of 116bp is amplified by using a recombinant plasmid pFB-HPV33L1 as a gene template and using primers F2 and R2, wherein a primer sequence F2 is shown as SEQ ID No. 10, and R2 is shown as SEQ ID No. 11.

The gene fragment contains a gene fragment of 31 (469-499) amino acids at the C end of HPV33L1, 10bp bases overlapped with a gene fragment of 1-467 amino acid C end of HPV56L1 and an XbaI (T ^ CTAGA) enzyme cutting site, and the amplified sequence is shown as SEQ ID No: 12.

PCR splicing sequence:

splicing primers are F1 and R2 respectively, and fragments obtained by amplifying the primers (fragments obtained by amplifying F1 and R1, fragments obtained by amplifying F2 and R2) are used as templates.

PCR splicing parameters: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 15s, annealing at 72 ℃ for 1kb/1min, and performing 5 cycles; denaturation at 98 ℃ for 10s, annealing at 68 ℃ for 15s, annealing at 72 ℃ for 1kb/1min, and performing 25 cycles; extending for 5min at 72 ℃; and finishing at 16 ℃.

Finally, SEQ ID NO 4 was obtained, which encodes a nucleotide sequence consisting of 1-467 amino acids of HPV56L1 and 31 (469-499) amino acids at the C-terminus of HPV33L1, with KpnI and XbaI cleavage sites at both ends (hereinafter referred to as "splicing sequence").

Double digestion of pFastBac with KpnI + XbaI^TM1 vector and splicing sequence fragment, cloning the splicing sequence to pFastBac^TM1 vector to obtain recombinant plasmid pFB-HPV56L 1: 33C. Namely the chimeric gene of replacing the C end of HPV56L1 with the C end of HPV33L 1.

Example 2: HPV56L 1:33C recombinant baculovirus packaging

pFB-HPV56L1 constructed in example 1:33C recombinant plasmid is identified and sequenced correctly, and then is transformed into DH10Bac bacteria competent cell (b)

The kit is purchased from Thermo Fisher), cultured and amplified at 37 ℃, subjected to plate streak culture, selected and amplified by white bacterial plaque, cultured overnight, collected bacterial liquid, and extracted by an alkaline lysis method to obtain recombinant bacmid DNA.

It was transfected into insect cells SF9 with cationic transfection reagent (purchased from nano Biological) for recombinant baculovirus seed packaging. The specific operation is as follows:

a. taking SF9 cells in logarithmic growth phase according to 0.6X 10⁶cell/dish Density inoculation dish, will be inoculated with SF9 cells dish room temperature 2h, adherence.

b. The extracted plasmid 20. mu.L Bacmid DNA was added to 200. mu.L Grace's Medium (serum-free, additive-free, from Gibico) and mixed and inverted 5 times.

c.25 μ L of 0.2 XTF 1 (transfection reagent, available from Sino Biological) was added dropwise to 200 μ L of Grace's Medium and gently mixed.

d. Mixing b and c. Incubating at room temperature for 15-45 min.

e. When the DNA was incubated with cellfectin (purchased from Sino Biological), the cell supernatant was discarded and Grace Medium with serum-free additives was added at 0.8 mL/dish.

f. The incubated DNA in d and transfection reagent complex was added dropwise to dish.

g.27 ℃ for 2 hr.

h. Cell culture was discarded and 2.5mL/dish complete growth medium (SCD6 SF + 10% FBS) (SCD6 SF from Sino Biological and FBS from Gibico) was added.

The cells were incubated at i.27 ℃ for 7 days to see if there was a viral infection.

After transfection, virus supernatant is collected after the cells have developed significant lesions and cultured for 7-11 days. Aseptically collecting the virus supernatant by using a pipette, namely obtaining HPV56L 1:33C P1 substitute for virus. Using HPV56L 1: the 33C P1 seed substitute infects SF9 cells according to the ratio of 1:50(V/V), and the infection density of SF9 cells is 2X 10⁶cells/mL, culturing and amplifying for 3 days at 27 ℃, centrifuging for 10min at the room temperature of 1000g +/-200 g, and collecting the virus supernatant, namely the P2 generation virus, which can be used for infection production.

Example 3: HPV56L 1:33C expression production

Using the HPV 56-containing L1 obtained in example 2: infecting High Five cells with baculovirus of 33C recombinant gene at an infection ratio of 1:200(V/V) at room temperature of 1000g +/-100 g, centrifuging at room temperature to collect cell precipitate, ultrasonically cracking the cell precipitate by using PBS or MOPS buffer solution (pH6.0-7.0, salt concentration of 100 mM-1M), ultrasonically crushing at low temperature for 3min, centrifuging at a centrifugal force of more than 10000g for 10min, collecting supernatant after centrifugation, and detecting by SDS-PAGE electrophoresis. Lane 1 Marker (Marker is 7 purified proteins with molecular weight fraction of 14.4 to 116kDa, from Thermo Scientific); lane 2: a cell lysate; lane 3: supernatant collected after centrifugation of the lysate.

The results are shown in FIG. 1, HPV56L1 prepared by this method: the 33C L1 protein yield is more than 100mg/L, the protein size is about 56KD, and the method can be used for large-scale production.

Example 4: HPV56L 1: purification preparation of 33C Virus-like particles

HPV56L 1: the purification method of 33C virus-like particles was a two-step chromatography, namely HS-MMA, and the supernatant collected in example 3 was purified to obtain virus-like particles of high purity.

The first step of chromatography:

medium: produced by Thermo Fisher corporation

50HS strong cation exchange media.

Volume of medium: the medium volume was 150mL and the linear flow rate was 30 mL/min.

Chromatography conditions are as follows: equilibration buffer (ph6.2, salt concentration 50mM phosphate, 0.5M sodium chloride); washing buffer (salt concentration 50mM phosphate, 0.75M sodium chloride, pH 6.2;)

The column was equilibrated with 5CV of buffer and then loaded. After the completion of the loading, the impure proteins were eluted with 5CV of equilibration buffer and washing buffer, respectively.

Elution conditions: the elution was carried out at pH6.2 with sodium chloride at an elution salt concentration of 1.25M using 50mM phosphate buffer containing 50mM arginine hydrochloride.

And a second step of chromatography:

medium: an MMA ion exchange medium produced by Shanghai Bogelong was used.

Chromatography conditions are as follows: equilibration buffer 50mM PB,1.25M NaCl, pH 6.2. The column was equilibrated with 4CV equilibration buffer before loading. After the loading is finished, the hybrid protein is washed by 5CV of balance buffer solution, and then the target protein is eluted by elution buffer solution to collect the protein.

Elution conditions: 100mM NaAC,150mM NaCl, 0.01% Tween 80, pH4.5.

Example 5: HPV56L 1: morphological examination of 33C virus-like particles

A10. mu.L sample was taken for transmission electron microscopy. Fixing the sample on a carbon copper-sprayed net for adsorption for 2min, absorbing residual liquid by using filter paper, dyeing twice by using phosphotungstic acid (Beijing Zhongjiekou Co., Ltd., concentration of 2%, pH6.5) for 30 seconds each time, absorbing residual dyeing liquid by using the filter paper, and observing under a transmission electron microscope after drying. The transmission electron microscope (brand: Hitachi, model: H-7650) was 80KV at 80,000 times magnification. As shown in FIG. 2, the result of electron microscope observation shows that HPV56L 1:33C modified at C-terminal can form virus-like particles with uniform size, and the average diameter is about 60 nm.

Example 6: HPV56L 1: evaluation of immunogenicity of 33C Virus-like particles in animals

6.1 modeling of pseudovirus-neutralizing cells

Since HPV is difficult to be cultured in vitro and has strong host specificity, it is difficult to be propagated in organisms other than human bodies, and a suitable animal model is lacking. Therefore, it is necessary to establish a suitable and effective in vitro neutralization experimental model for the evaluation of vaccine immunoprotection.

The HPV pseudovirus is an ideal HPV in vitro neutralization experimental model: by utilizing the characteristic that HPV VLP has non-specific encapsulated nucleic acid, VLP consisting of HPV L1 and L2 expressed in cells is encapsulated with free DNA or introduced into exogenous plasmid to form HPV pseudovirus.

And (3) performing immunogenicity analysis on the animal serum sample after the sample is immunized by adopting a pseudovirus neutralization method. After the HPV56 virus-like particle sample is used for immunizing animals, neutralizing antibodies against HPV56 can be generated, and pseudoviruses of HPV56 type can be neutralized. After the immune animal serum is incubated with a certain amount of pseudovirus and then infected with cells, the cells capable of expressing GFP fluorescence can be reduced along with the increase of neutralizing antibodies in the serum, and linear negative correlation can be formed in a certain range, so that the neutralizing activity of the antibodies in the serum can be evaluated by detecting the change of the number of the cells expressing GFP.

The pseudovirus construction method comprises the following steps: pCMV3-3-HPV56L1+ L2(L1 sequence from Uniprot P36743, L2 sequence from Uniprot P36765) plasmid (from Sino Biological) and fluorescent plasmid (PSEU-GFP Spark, from Sino Biological) of HPV type 56 were co-transfected into 293FT adherent cells (from Thermo Fisher). Specific process references (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.reaction of human sera in a sensitive, high-throughput pseudomonas-based mammalian lysis assay for HPV16 and HPV18.[ J ] Virology 2004,321: 205-. Collecting pseudovirus supernatant, subpackaging, and storing in a refrigerator at-80 deg.C for use.

6.2 HPV56L 1:33C virus-like particle animal immunoprotection evaluation

Mouse immunization procedure:

HPV56L 1: adsorbing the 33C virus-like particles on an aluminum phosphate adjuvant, mixing, taking 200 mu L of the mixture to immunize mice, wherein the immunization dose of each mouse is 0.15 mu g, immunizing 10 mice, immunizing the mice with diluted samples respectively on the 0 th day, the 7 th day and the 21 st day of an experiment, simultaneously establishing a blank serum control group, taking blood from eyeballs of the mice on the 28 th day of the experiment, and separating serum to carry out pseudovirus neutralization titer detection.

Mouse EC50 assay:

mouse serum was inactivated at 56 ℃ for 30 minutes, centrifuged at 6000g, and the supernatant was assayed after 5 minutes. 293FT cells were plated at a density of 15000 cells/well in 96-well plates and cultured at 37 ℃ with 5% C4-8 hours prior to assayO ₂In a carbon dioxide incubator. After immunization, the mouse serum and the blank control serum are serially diluted by a neutralization medium and are respectively mixed with the HPV56 pseudovirus prepared in 6.1 according to the volume ratio of 1: 1. And incubating for 1 hour in a refrigerator at the temperature of 2-8 ℃, adding the mixture to 293FT cells plated 4-8 hours in advance according to 100 mu L/hole, and setting a blank serum control hole, a pseudovirus positive control hole and a negative control hole in 2 duplicate holes for each sample. The loaded cells were continued at 37 ℃ with 5% CO₂After culturing in the carbon dioxide incubator of (1) for 62 to 96 hours, a photograph was taken by fluorescent scanning and counted in an enzyme-linked spot Analyzer (model: S6 Universal-V Analyzer, manufacturer: CTL). Calculating the neutralization inhibition rate of each mouse serum sample, and calculating the maximum dilution factor of the serum when the neutralization inhibition rate of the serum is 50 percent according to a Reed-Muench method, namely the half effective dilution factor EC₅₀。

The results of the HPV56 serum pseudovirus neutralization titer test are detailed in Table 4.

TABLE 4 mouse serum neutralization titre assay results EC₅₀(GMT±SEM)

Remarking:

1. number of animals, N ═ 10;

GMT (geometric Mean titer): geometric mean titer;

SEM (Standard Error of mean): standard error.

The detection result shows that the HPV56L1 prepared by the invention has the following characteristics: the 33C virus-like particle has better immunogenicity, can generate high-titer neutralizing antibodies in animal bodies, and can be used for preparing vaccines for preventing HPV infection.

Comparative example 1: expression of C-terminally truncated HPV16L1(1-474)

The inventors tried to truncate the C-terminus of HPV16L1 by 31 amino acids, named HPV16L1(1-474) (SEQ ID NO: 14). However, in the research, the truncated HPV16L1(1-474) has high protein expression amount and poor protein solubility, and is difficult to extract and purify, and the specific expression and extraction results are shown in FIG. 3.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that various changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Appendix 1: sequence listing

Claims

A chimeric HPV type 56L1 protein comprising, in the N-terminal to C-terminal direction:

a. an N-terminal fragment derived from an HPV type 56L1 protein, said N-terminal fragment retaining the immunogenicity of the HPV type 56L1 protein; and

b. a C-terminal fragment derived from a second type papillomavirus L1 protein, said second type papillomavirus L1 protein having the property of better expression and solubility than other types of L1 protein;

wherein the chimeric HPV type 56L1 protein has the immunogenicity of an HPV type 56L1 protein.
The chimeric HPV type 56L1 protein according to claim 1, wherein

The N-terminal fragment is a fragment obtained by cutting the C-terminal of the natural sequence of the HPV56 type L1 protein shorter than any amino acid position in the alpha 5 region of the protein, and the fragment has at least 98 percent of identity with the fragment; and is

The C-terminal fragment is a fragment obtained by cutting off the N-terminal of the natural sequence of the second type papilloma virus L1 protein to be shorter than any amino acid position in the alpha 5 region thereof, and a functional variant generated by further mutating, deleting and/or adding the fragment.
The chimeric HPV type 56L1 protein of claim 2, wherein the C-terminal fragment comprises one or more nuclear localization sequences.
The chimeric HPV type 56L1 protein according to claim 1, wherein the second type papillomavirus L1 protein is selected from HPV type 1, type 2, type 3, type 4, type 6, type 7, type 10, 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, type 44, type 45, type 51, type 52, type 53, type 56, type 58, type 59, type 60, type 63, type 66, type 68, type 73 or type 82L 1 protein;

preferably, the second type papillomavirus L1 protein is selected from HPV type 16, type 28, type 33, type 59, or type 68L 1 protein;

more preferably, said second type papillomavirus L1 protein is selected from the HPV type 33 or HPV type 59L 1 proteins.
The chimeric HPV type 56L1 protein according to claim 4 wherein the C-terminal fragment is SEQ ID No 2; or a fragment thereof of length m amino acids, preferably a fragment encompassing amino acids 1-m of SEQ ID No. 2; wherein m is an integer of 13 to 31.
The chimeric HPV type 56L1 protein according to claim 4 wherein the C-terminal fragment is SEQ ID No 13; or a fragment thereof of length n amino acids, preferably a fragment encompassing amino acids 1 to n of SEQ ID No. 13; wherein n is an integer from 16 to 38.
The chimeric HPV type 56L1 protein according to claim 1, wherein the N-terminal fragment is 98%, 98.5%, 99%, 99.5% or 100% identical to a fragment obtained by truncating the C-terminal end of the sequence shown in SEQ ID No:1 shorter than any amino acid position within its alpha 5 region.
The chimeric HPV type 56L1 protein according to claim 1, the C-terminus of the N-terminal fragment being linked to the N-terminus of the C-terminal fragment either directly or through a linker.
The chimeric HPV type 56L1 protein according to claim 1, wherein when the C-terminus of the N-terminal fragment is linked to the N-terminus of the C-terminal fragment there is the following contiguous amino acid sequence within plus or minus 4 amino acid positions of the point of linkage: RKFL;

preferably, the following contiguous amino acid sequence is present within plus or minus 6 amino acid positions of the point of attachment: LGRKFL.
The chimeric HPV type 56L1 protein according to claim 1 having 98%, 98.5%, 99%, 99.5% or 100% identity to SEQ ID No 3.
A virus-like particle of HPV type 56 comprising the chimeric HPV type 56L1 protein of any one of claims 1-10.
The HPV type 56 virus-like particle of claim 11, which is an icosahedron consisting of a pentamer of 72 of the chimeric HPV type 56L1 protein.
An immunogenic composition for the prevention of an HPV-related disease or infection comprising a HPV type 56 virus-like particle of claim 11 or 12 and an adjuvant.
An isolated polynucleotide encoding the chimeric HPV type 56L1 protein of any one of claims 1-10.
An isolated polynucleotide having the sequence shown in SEQ ID No. 4.
A vector comprising the polynucleotide of claim 14 or 15.
The vector of claim 16, wherein the vector is a baculovirus vector.
A baculovirus comprising the polynucleotide of claim 14 or 15.
A host cell comprising the polynucleotide of claim 14 or 15, the vector of claim 16 or 17, or the baculovirus of claim 18.
The host cell according to claim 19, which is an insect cell, preferably the insect cell is selected from the group consisting of Sf9 cell, Sf21 cell, Hi5 cell and S2 cell.
A method of making the HPV type 56 virus-like particle of claim 11 or 12, comprising:

culturing the host cell of any one of claims 19 to 20 to express the chimeric HPV type 56L1 protein and assemble into a virus-like particle; and

purifying the HPV type 56 virus-like particle.
The method of claim 21, wherein the host cell is a Hi5 cell.
The process according to claim 21, wherein the purification employs cation exchange chromatography, preferably the cation exchange chromatography is HS-MMA two-step chromatography.