CN102747047A - Human papillomaviruse type hybrid virus-like particles and preparation method thereof - Google Patents
Human papillomaviruse type hybrid virus-like particles and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to human papillomavirus (HPV) type hybrid virus-like particles and a preparation method thereof. The virus-like particles can be used for preventions two or more HPV infections and diseases caused by HPV infections. The present invention further relates to uses of the protein and the virus-like particles in preparations of drug compositions or vaccines, wherein the drug compositions or the vaccines are provided for preventions of HPV infections and diseases caused by HPV infections, and the diseases comprise cervical cancer, condyloma acuminatum, and the like.
Description
Technical field
The present invention relates to Molecular Virology and field of immunology.Particularly; The present invention relates to the other hybrid virus appearance of human papillomavirus type particle that is formed by different type HPV L1 albumen heterozygosis assemblings and preparation method thereof, said type hybrid virus appearance particle can be used for preventing two or more HPV to infect and reaches the disease that is caused by the HPV infection.The invention still further relates to above-mentioned type hybrid virus appearance particle and be used for the purposes of pharmaceutical compositions or vaccine, said pharmaceutical composition or vaccine are used to prevent HPV to infect and are infected disease such as cervical cancer, the pointed condyloma etc. that caused by HPV.
Background technology
(Human Papillomavirus HPV) mainly causes skin to human papillomavirus, the excipuliform pathology of mucous membrane.According to itself and tumorigenic relation, HPV can be divided into high-risk-type and low risk, and wherein the HPV of high-risk-type infects to be proved to be and brings out the anus sexual organ cancer major cause that comprises women's cervical cancer; Low risk then mainly causes pointed condyloma.The effective means that prevention and control HPV infect is the HPV vaccine, particularly can cause the high-risk HPV vaccine of cervical cancer.
It is hollow virus-like particle (Virus-Like Particle, characteristic VLP) that the main capsid protein L 1 of HPV has self-assembly.HPV VLP is connected with disulfide linkage and is constituted the three-dimensional symplex structure of 20 bodies (Doorbar, J.and P.H.Gallimore.1987.J Virol, 61 (9): 2793-9) by the pentamer of 72 main capsid protein L 1s.HPV VLP structure is highly similar with natural HPV, has kept most neutralizing epitopes of natural viral, can induce neutralizing antibody (Kirnbauer, R., F.Booy, the et al.1992Proc Natl Acad Sci U S A 89 (24): 12180-4) of high titre.Structural research and mutation analysis show; HPV16 L1 PROTEIN C ys175, Cys185 and Cys428 play a key role to the interaction between the fundamental structural unit pentamer of VLP; Cys161, Cys229, Cys379 are essential for the interaction between monomer molecule; Prompting occur in the L1 protein molecular and/or intermolecular disulfide linkage for the important effect of being assembled with of HPV virus-like particle (Kanda is Virology.308:128-136 T.2003 for Ishii Y., Tanaka K.).This research is promptly transformed these disulfide linkage, invents new artificial HPV type hybrid virus appearance particle.
Existing discovering, HPV VLP mainly induces the neutralizing antibody to homotype HPV, produces the protective immunity to homotype HPV, only between the high type of some homologys, has cross protection.Therefore, the protection domain of existing HPV vaccine is limited.Can only realize through increasing the HPV VLP type that is contained in the vaccine if enlarge the protection domain of HPV vaccine.This will improve the production cost of HPV vaccine greatly, and possibly bring the potential safety issue because immunizing dose increases.And the HPV vaccine that has gone on the market at present (comprising:
HPV16 of Merck company; 18; 6;
HPV16 of 11 tetravalent vaccines and GSK company, 18 bivalent vaccines) all adopt independent type VLP to be mixed with to form.Therefore, this area still needs to induce the HPV L1 virus-like particle to many types of other HPV protection antibody, thereby makes the cervical cancer vaccine of the more many types of other HPV of preparation prevention become possibility.
Summary of the invention
The present invention at least part based on contriver's following beat all discovery: utilize escherichia expression system to express the HPV L1 albumen of other 175 of two or more different shaped and 428 cysteine mutation or disappearance; The HPV L1 albumen of said sudden change or disappearance can not form virus-like particle separately; Yet; Mix the back according to certain hybrid mode and certain concentration ratio and remove reductive agent, can obtain a kind of new heterozygosis VLP.This heterozygosis VLP contains two or more HPV L1 albumen, can induce the high titre neutralizing antibody to two or more HPV.
Therefore; On the one hand; The present invention relates to be assembled into the HPV L1 albumen of sudden change of hybrid virus appearance particulate or disappearance, said albumen includes but not limited to: HPV L1-C175A, HPV L1-Δ C428, HPV L1-Δ C175, HPV L1-C175S, HPV L1-C428A, HPV L1-C428S etc.
In a preferred embodiment, HPV L1-C175A and HPV L1-Δ C428 were according to 1: 1 mixed in molar ratio.In other embodiments, the ratio of the two comprise 2: 1,1: 2 or the like.
In yet another aspect, the present invention relates to a kind of preparation hybrid virus appearance of the present invention particulate method.In a preferred embodiment, preparing hybrid virus appearance particulate method of the present invention comprises:
A) at said sudden change of expression in escherichia coli or disappearance HPV L1 albumen,
B) will express said sudden change or disappearance HPV L1 protein purification, obtain the sudden change or the disappearance HPV L1 albumen of purity more than 95%,
C) with b) sudden change of purifying or disappearance L1 albumen: HPV L1-C175A and HPV L1-Δ C428 were according to 1: 1 mixed in molar ratio, and dialysis removal reductive agent promptly obtains heterozygosis VLP.
D) with c) the heterozygosis VLP that obtains is further purified the collecting granules component.
The invention still further relates to a kind of method for preparing vaccine; It comprises HPV heterozygosis VLP of the present invention and pharmaceutically acceptable carrier and/or mixed with excipients, randomly also mixes one or more virus-like particles that are selected from HPV6,11,16,18,31,33,45,52,58 and 59 HPV type or the heterozygosis VLP of other types.As above discussed, the vaccine that is obtained can be used to prevent HPV to infect or infect the disease that caused such as cervical cancer etc. by HPV.
In yet another aspect, also relate to according to the purposes of hybrid virus appearance particle of the present invention in pharmaceutical compositions or vaccine, said pharmaceutical composition or vaccine are used to prevent HPV to infect or are infected the disease that is caused by HPV.
The explanation of relational language and explanation among the present invention
In the present invention, except as otherwise noted, otherwise the Science and Technology noun that uses among this paper has the implication of those skilled in the art institute common sense.And cell cultures, molecular genetics, nucleic acid chemistry, Immunology Lab operation steps used among this paper are widely used conventional steps in the corresponding field.Simultaneously, in order to understand the present invention better, the definition and the explanation of relational language are provided below.
According to the present invention, term " HPV L1 " is meant the L1 albumen of human papillomavirus (HPV), and its sequence is well known in the art (referring to, ncbi database sequence number for example: DQ469930.1).In the present invention, when relating to the sequence of HPV L1, with reference to Ishii Y, Tanaka K, Kanda T.Virology. (2003), 308 (1), 128-36.It uses the ncbi database sequence number: DQ469930.1 describes.
According to the present invention; Term " HPV L1 mutant protein " is meant; A certain position in L1 albumen or a few halfcystines (C) residue lacks or with the protein that other radical amino acid replacement obtained not necessarily comprises L1 albumen is carried out the disappearance that N end or C hold.
According to the present invention; Term " HPV X-C Y Z " is meant; Halfcystine (C) protein that residue obtained with Z radical amino acid replacement HPV X type L1 protein Y position; Wherein X is meant the type of HPV; Y be meant GenBank ABF06542.1 (its nucleotides sequence is classified DQ469930.1 as) aminoacid sequence from the residing position of amino acid that N end methionine(Met) (M) begins to count, like X=16, Y=175 is meant the 175th amino acids halfcystine C of ABF06542.1 aminoacid sequence; After the Y value of non-HPV16 type then refers to target protein sequence and ABF06542.1 aminoacid sequence carried out sequence alignment (can use Blast, Fasta, Cluster supervisor), corresponding to the position of ABF06542.1 aminoacid sequence.Refer to the 175th halfcystine (C) protein that residue obtained like " HPV16-C175A " with L-Ala (A) residue displacement HPV16 type L1 a-protein BF06542.1; " HPV52-C175A " refers to the protein that obtained corresponding to cysteine residues (C) back of the 175th residue of ABF06542.1 aminoacid sequence with in L-Ala (A) the residue displacement HPV52 type L1 protein.
According to the present invention, term " HPV X-Δ C Y " is meant, with the protein that halfcystine (C) the residue disappearance of the proteinic Y of HPV X type L1 position is obtained, wherein the amino acid position numbering is suitable for above definition.Refer to the 428th protein that cysteine residues disappearance back is obtained like " HPV16-Δ C428 " with HPV16 type L1 a-protein BF06542.1; " HPV52-Δ C428 " refers to the protein that obtained corresponding to cysteine residues (C) the disappearance back of the 428th residue of ABF06542.1 aminoacid sequence in the HPV 52 type L1 protein.
According to the present invention, term " heterozygosis particle " is meant, type more than 2 kinds or 2 kinds can not be assembled the L1 albumen that forms VLP separately mix assembling, formed VLP after sudden change.
According to the present invention, term " X and Y heterozygosis VLP " is meant, X is mixed assembling with Y albumen, formed heterozygosis VLP.Be meant HPV16 L1-C175A is mixed assembling, formed heterozygosis VLP with HPV52 L1-Δ C428 albumen like " HPV16 L1-C175A and HPV52 L1-Δ C428 heterozygosis VLP ".
According to the present invention, term " variant " is meant such albumen, and its aminoacid sequence has one or more (for example 1-10 or 1-5 or 1-3) amino acid different (for example conservative amino acid replacements) or has at least 60% with the proteic aminoacid sequence of HPV L1 of sudden change of the present invention; 80%, 85%, 90%; 95%, 96%, 97%; 98%, or 99% identity, and it has kept the necessary characteristic of said truncated protein.Term " necessary characteristic " can be one or more in the following characteristic here: can induce the neutralizing antibody to this type HPV; Can in intestinal bacteria, express on solubility ground; Utilize expression and purification method involved in the present invention can obtain the purifying protein of high yield.
According to the present invention, term " identity " be used in reference between two polypeptide or two nucleic acid between the match condition of sequence.When certain position in two sequences that compare is all occupied by identical base or amino acid monomer subunit (for example; Certain position in each of two dna moleculars is all occupied by VITAMIN B4; Or certain position in each of two polypeptide is all occupied by Methionin), each molecule is same on this position so." percentage ratio identity " between two sequences is by the function of the total matched position number of these two sequences divided by position number * 100 that compare.For example, if in 10 positions of two sequences 6 couplings are arranged, these two sequences have 60% identity so.For example, dna sequence dna CTGACT and CAGGTT have 50% identity (in 6 positions 3 location matches being arranged altogether).Usually, two sequence alignments are being compared when producing maximum identity.Such comparison can be through using, for example, can through computer program for example the Align program (DNAstar, the method for people such as Needleman (1970) J.Mol.Biol.48:443-453 that Inc.) carries out easily realizes.Also can use the E.Meyers and W.Miller (the Comput.Appl Biosci. of the ALIGN program of having integrated (version 2 .0); 4:11-17 (1988)) algorithm uses PAM120 weight residue table (weight residue table), 12 notch length point penalty and 4 breach point penalty to measure two percentage ratio identity between the aminoacid sequence.In addition; Can use Needleman and Wunsch (J MoI Biol.48:444-453 (1970)) algorithm in the GAP program that has been integrated into GCG software package (can on www.gcg.com, obtain), use Blossum 62 matrixes or PAM250 matrix and 16,14,12,10,8,6 or 4 breach weight (gap weight) and 1,2,3,4,5 or 6 length weight to measure two percentage ratio identity between the aminoacid sequence.
Like what use among this paper, term " conservative substitution " means the amino-acid substitution that can influence sharply or change the BA of the albumen/polypeptide that comprises aminoacid sequence.For example, can for example site-directed mutagenesis and PCR mediated mutagenesis be introduced conservative substitution through standard technique known in the art.Conservative amino acid replacement comprises the displacement that substitutes amino-acid residue with the amino-acid residue with similar side chain; For example be used on the physics or displacement that the residue of similar with corresponding amino-acid residue on the function (for example have similar size, shape, electric charge, chemical property, comprise the ability that forms covalent linkage or hydrogen bond etc.) carries out.In this area, defined the family of amino-acid residue with similar side chain.These families comprise (for example having basic side chain; Methionin, l-arginine and Histidine), acid side-chain (for example aspartic acid, L-glutamic acid), uncharged polar side chain (for example glycocoll, l-asparagine, Stimulina, Serine, Threonine, tyrosine, halfcystine, tryptophane), non-polar sidechain (for example L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine(Phe), methionine(Met)), β branched building block (for example; Threonine, Xie Ansuan, Isoleucine) and the amino acid of aromatic series side chain (for example, tyrosine, phenylalanine(Phe), tryptophane, Histidine).Therefore, preferably use another amino-acid residue to substitute corresponding amino-acid residue from same side chain family.The method of identifying conservative aminoacid substitutions in this area be know (referring to, for example, people such as Brummell, Biochem.32:1180-1187 (1993); People Protein Eng.12 (10): 879-884 (1999) such as Kobayashi; With people Proc.Natl Acad.Set USA94:412-417 (1997) such as Burks, it incorporates this paper by reference into).
According to the present invention; Term " escherichia expression system " is meant the expression system of being made up of intestinal bacteria (bacterial strain) and carrier; Wherein intestinal bacteria (bacterial strain) derive from available bacterial strain on the market, such as but not limited to: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3) or the like.
According to the present invention, term " carrier (vector) " is meant, can polynucleotide be inserted a kind of nucleic acid vehicle wherein.When carrier can make the coded albumen of polynucleotide of insertion obtain to express, carrier was called expression vector.Carrier can be through transforming, and transduction or transfection import host cell, makes its genetic material element that carries in host cell, obtain to express.Carrier is well known to a person skilled in the art, includes but not limited to: plasmid; Phage; Coemid or the like.
The damping fluid that can be used in the method for the present invention is well known in the art, includes but not limited to Tris damping fluid, phosphate buffered saline buffer, HEPES damping fluid, MOPS damping fluid or the like.
Can obtain through following steps according to HPV hybrid virus appearance particle of the present invention: the sudden change of above-mentioned purity at least 95% or the HPV L1 albumen of disappearance are assembled by 1: 1 mixed in molar ratio; Remove the reductive agent in this solution, obtain said HPV hybrid virus appearance particle.The mode of removing reductive agent is known in the art, includes but not limited to dialysis, ultrafiltration or chromatography etc.
According to the present invention; Term " pharmaceutically acceptable carrier and/or vehicle " is meant carrier and/or vehicle compatible with activeconstituents with the experimenter on pharmacology and/or physiology; It is well known in the art (referring to for example Remington ' s Pharmaceutical Sciences.Edited by Gennaro AR, 19th ed.Pennsylvania:Mack Publishing Company, 1995); And include but not limited to: the pH regulator agent; Tensio-active agent, adjuvant, ionic strength toughener.For example, the pH regulator agent includes but not limited to phosphate buffered saline buffer; Tensio-active agent comprises but is not limited to positively charged ion, negatively charged ion or non-ionics, for example Tween-80; Adjuvant includes but not limited to aluminium adjuvant (for example white lake), freund's adjuvant (for example complete Freund's adjuvant); The ionic strength toughener includes but not limited to sodium-chlor.
The beneficial effect of the invention
Preparation HPV virus-like particle is the VLP that single type L1 assembling forms at present, mainly induces the neutralizing antibody to homotype HPV, produces the protective immunity to homotype HPV, only between the high type of some homologys, has cross protection.Therefore, the protection domain of existing HPV vaccine is limited.Can only realize through increasing the HPV VLP type that is contained in the vaccine if enlarge the protection domain of HPV vaccine.This will improve the production cost of HPV vaccine greatly, and possibly bring the potential safety issue because immunizing dose increases.
Though there are some researches show; HPV L1 proteic 175 and 428 halfcystines maybe its vital role for the virus-like particle assembling; Sudden change or delete the formation that these amino acid possibly influence HPVVLP; But the present invention gropes through mutation analysis and assembling condition, finds unexpectedly that but these halfcystines only influence type HPV L1 protein groups of the same race and dress up VLP; But this influence but can be eliminated through different type HPV L1; Thereby the method for the different type HPV L1 assembling of invention preparation heterozygosis VLP, this method has been got rid of type HPV L1 protein groups of the same race fully and has been dressed up the possibility into VLP, because proteic 175 or 428 two mutants self of the HPV L1 of type can't form VLP separately; Have only and the complementary mutually VLP that could form heterozygous of the HPV L1 protein mutant of other types; Such method not only can be applied to structure and the assembly mechanism research of HPV VLP, and the purposes of vaccine production, also can be applicable to based on the nano material of HPV VLP structure and assembling and relevant research.
It is good that different type L1 albumen heterozygosis assembling formation heterozygosis are had immunogenicity; Be prone to produce the advantages such as cross protection of non-vaccine type, but as adopting the direct combined group of different type L1 albumen to dress up VLP, not only packaging efficiency is low; The VLP form that is assembled into is also relatively poor; Also exist simultaneously possibly to have various proportionings and the inhomogenous problem of VLP has limited the industrialized preparation of heterozygosis VLP, had a strong impact on the immunogenicity of the heterozygosis VLP that obtains.
Heterozygosis VLP provided by the invention and its preparation method have effectively solved the problems referred to above.At first, it is raw material that the present invention adopts the L1 albumen that can't be assembled into VLP after the sudden change separately, has guaranteed that it is easy to preparation.Secondly, with certain condition and ratio assembling preparation heterozygosis VLP, can obtain the heterozygosis VLP of high packaging efficiency, and the assembling ratio is consistent, the heterozygosis VLP state homogeneous that assembling obtains with this mutain.Further, this heterozygosis VLP can obtain the neutralizing antibody of higher titre in vivo than VLP mixed immunity of the same race under Isodose, have better immunogenicity; In addition; Can also obtain the neutralizing antibody of the non-vaccine type of higher titre, VLP compares with the blended multivalence, is more suitable for using as vaccine.
To combine accompanying drawing and embodiment that embodiment of the present invention are described in detail below, but it will be understood by those skilled in the art that attached drawings and embodiment only are used to explain the present invention, rather than to the qualification of scope of the present invention.According to the following detailed description of accompanying drawing and preferred embodiment, it is obvious that various purposes of the present invention and favourable aspect will become to those skilled in the art.
Description of drawings
Fig. 1 has shown the result of the sds polyacrylamide gel electrophoresis of the HPV-L1 two mutants that obtains through HIC (hydrophobic interaction chromatograph) purifying among the embodiment 1.Swimming lane M: molecular weight of albumen mark; Swimming lane 1:HPV16L1-C175A is through HIC (hydrophobic interaction chromatograph) purifying elutriated fraction; Swimming lane 2:HPV16L1-Δ C428 is through HIC (hydrophobic interaction chromatograph) purifying elutriated fraction; Swimming lane 3:HPV52L1-C175A is through HIC (hydrophobic interaction chromatograph) purifying elutriated fraction; Swimming lane 4:HPV52L1-Δ C428 is through HIC (hydrophobic interaction chromatograph) purifying elutriated fraction; The result shows that behind Butyl Sepharose 4 Fast Flow column purification, HPV16L1-C175A, HPV16L1-Δ C428, HPV52L1-C175A, HPV52L1-Δ C428 purity of protein reach more than 95%.
Fig. 2 has shown transmission electron microscope observing (amplifying 100,000 times, Bar=0.1 μ m) result after the HPV-L1 mutant protein renaturation of gained among the embodiment 2.Fig. 2 A, the HPV16L1-C175A albumen after the renaturation; Fig. 2 B, the HPV16L1-Δ C428 albumen after the renaturation; Fig. 2 C, the HPV52L1-C175A albumen after the renaturation; Fig. 2 D, the HPV52L1-Δ C428 albumen after the renaturation.The result shows that the visible a large amount of pentamers of radius about 5nm do not have virus-like particle to exist in the visual field of these 4 figure.
The HPV16 L1-C175A that Fig. 3 has shown method described in the embodiment 3 preparation and HPV52L1-Δ C428 under different buffer conditions, the transmission electron microscope observing result (100,000 times, Bar=0.1 μ m) of the heterozygosis VLP that assembled in 1: 1 in molar ratio.Fig. 3 A, HPV16L1-C175A and HPV52L1-Δ C428 are at 50mM PB (sodium phosphate buffer), pH6.0, the heterozygosis VLP that 0.5M NaCl is assembled into; Fig. 3 B, HPV16 L1-C175A and HPV52L1-Δ C428 be at 20mM PB, pH6.5, the heterozygosis VLP that 0.5M NaCl is assembled into; Fig. 3 C, HPV16 L1-C175A and HPV52L1-Δ C428 be at 20mM PB, pH7.0, the heterozygosis VLP that 0.5M NaCl is assembled into.The result shows; Visible big or small heterogeneity radius is the virus-like particle about 20nm in Fig. 3 A visual field, visible big or small homogeneous in Fig. 3 B visual field, the virus-like particle about radius 30nm; The visible a large amount of pentamers of radius about 5nm do not have virus-like particle to exist in Fig. 3 C visual field.
Fig. 4 has shown the HPV16 L1-C175A and the result of HPV52 L1-Δ C428 by the gel permeation chromatography of the heterozygosis VLP of different molar ratio assemblings of the preparation of method described in the embodiment 4.The result shows that the heterozygosis VLP particle assembling ratio of HPV16 L1-C175A and HPV52L1-Δ C428 assembling in 1: 1 in molar ratio is more suitable, and the pentamer residue is minimum.
Fig. 5 has shown the HPV16 L1-C175A and the transmission electron microscope observing result (100,000 times, Bar=0.1 μ m) of HPV52 L1-Δ C428 by the heterozygosis VLP of different molar ratio assemblings of the method preparation described in the embodiment 4.Fig. 5 A, the heterozygosis VLP of HPV16 L1-C175A and HPV52L1-Δ C428 assembling in 2.5: 1 in molar ratio; Fig. 5 B, the heterozygosis VLP of HPV16 L1-C175A and HPV52L1-Δ C428 assembling in 2: 1 in molar ratio; Fig. 5 C, the heterozygosis VLP of HPV16 L1-C175A and HPV52L1-Δ C428 assembling in 1: 1 in molar ratio; Fig. 5 D, the heterozygosis VLP of HPV16 L1-C175A and HPV52L1-Δ C428 assembling in 1: 2 in molar ratio.The result shows that the visible a large amount of pentamers of radius about 5nm do not have virus-like particle to exist in Fig. 5 A visual field.Visible big or small heterogeneity in Fig. 5 B visual field, the virus-like particle about radius 20-30nm; Visible big or small homogeneous in Fig. 5 C visual field, the virus-like particle about radius 30nm; Can be big or small inhomogenous in the visual field in Fig. 5 D visual field, the virus-like particle about radius 20nm, and the pentamer of a large amount of radiuses about 5nm arranged.
Fig. 6 has shown result and the three-dimensional structure of reconstruction thereof of the freezing electron microscopic observation of HPV16L1-C175A that embodiment 4 said methods obtain and HPV52L1-Δ C428 heterozygosis VLP.Fig. 6 A, HPV16L1-C175A and HPV52L1-Δ C428 heterozygosis VLP; Fig. 6 B, the three-dimensional structure of the reconstruction of HHPV16L1-C175A and HPV52L1-Δ C428 heterozygosis VLP.The three-dimensional structure of rebuilding shows, HPV16L1-C175A and HPV52L1-Δ C428 heterozygosis VLP be by the icosahedral structure of virus of the T=7 of 72 capsomeres (form subunit, pentamer) formation (h=1, k=2).Different with the general icosahedron viruses capsid that meets the quasi-equivalence principle, all composition subunits are pentamer in HPV16L1-C175A and the HPV52L1-Δ C428 heterozygosis VLP VLP structure, and do not see six aggressiveness, and the outermost diameter of VLP is about 60nm.This and three-dimensional structure (Baker TS, the Newcomb WW of the HPV VLP in natural HPV virion and eukaryotic expression system (for example, the pox viruses express system) source of report before; Olson NH.et al.Biophys J. (1991), 60 (6): 1445-1456.Hagensee ME, Olson NH; Baker TS, et al.J Virol. (1994), 68 (7): 4503-4505.Buck CB; Cheng N, Thompson CD.et al.J Virol. (2008), 82 (11): 5190-7.) similar.
Fig. 7 has shown the HPV16 L1-C175A of preparation among the embodiment 4 and the result of HPV52L1-Δ C428 heterozygosis VLP sds polyacrylamide gel electrophoresis.Swimming lane M: molecular weight of albumen mark; Swimming lane 1:HPV16L1 albumen; Swimming lane 2:HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP; Swimming lane 3:HPV52L1 albumen.The result shows: electrophoresis result shows that HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP have two specific bands, and is consistent with the band of HPV16L1, HPV52L1 respectively.
Fig. 8 has shown among the embodiment 4 that HPV16 L1-C175A and the HPV52L1-Δ C428 heterozygosis VLP of preparation and the special linear antibody 21A5 of HPV16 carry out the protein immunoblotting detected result.Swimming lane M: molecular weight of albumen mark; Swimming lane 1:HPV52L1 albumen; Swimming lane 2:HPV16L1 albumen; Swimming lane 3:HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP.The result shows that the special linear antibody 21A5 Western reaction of HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP and HPV52 has specific band.
Fig. 9 has shown among the embodiment 4 that HPV16 L1-C175A and the HPV52L1-Δ C428 heterozygosis VLP of preparation and the special linear antibody 12D10 of HPV52 carry out the protein immunoblotting detected result.Swimming lane M: molecular weight of albumen mark; Swimming lane 1:HPV52L1 albumen; Swimming lane 2:HPV16L1 albumen; Swimming lane 3:HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP.The result shows that the special linear antibody Western reaction of HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP and HPV52 has specific band.
Figure 10 has shown the HPV16 L1-C175A and the HPV52L1-Δ C428 heterozygosis VLP of preparation among the embodiment 4, the result of mutant protein and HPV VLP heat stability test.
Figure 11 has shown immune different steps serum NAT detected result among the embodiment 7.The result shows that the HPV16 L1-C175A and HPV52L1-Δ C428, HPV52L1-C175A and the HPV16L1-Δ C428 heterozygosis VLP that obtain according to the said method of embodiment 1-4 have good immunogenicity, can induce the neutralizing antibody to HPV16, HPV52 of high titre in the mouse body.Also produce simultaneously to HPV58,33,35 neutralizing antibody.
Figure 12 has shown HPV52L1-C175A described in the embodiment 5 and HPV16L1-Δ C428 heterozygosis VLP; HPV16L1-C175A and HPV6L1-Δ C428 heterozygosis VLP; HPV6L1-C175S and HPV16L1-Δ C428 HPV6 heterozygosis VLP, HPV6L1-C175S and HPV52L1-Δ C428 HPV52 L1-C175S, HPV52L1-C175A and the transmission electron microscope observing result (100 of HPV6L1-Δ C428 heterozygosis VLP in store buffer liquid; 000 times, Bar=0.1 μ m).Figure 12 A, HPV52L1-C175A and HPV16L1-Δ C428 heterozygosis VLP; Figure 12 B, HPV16L1-C175A and HPV6L1-Δ C428 heterozygosis VLP; Figure 12 C, HPV6L1-C175S and HPV16L1-Δ C428 HPV6 heterozygosis VLP; Figure 12 D, HPV6L1-C175S and HPV52L1-Δ C428 heterozygosis VLP; Figure 12 E, HPV52L1-C175A and HPV6L1-Δ C428 heterozygosis VLP.The result shows that all visible size is the virus-like particle about 25nm than the homogeneous radius in the visual field.
Embodiment
Existing the present invention is described with reference to the following embodiment that is intended to illustrate the present invention (and non-limiting the present invention).
Only if specialize, employed experimental methods of molecular biology and immunodetection among the present invention, basically with reference to people such as J.Sambrook, molecular cloning: laboratory manual; The 2nd edition, press of cold spring harbor laboratory, 1989; And people such as F.M.Ausubel, fine works molecular biology experiment guide, the 3rd edition; John Wiley & Sons, Inc., the method described in 1995 is carried out; The condition that the use of restriction enzyme is recommended according to the goods producer.Those skilled in the art know, and embodiment describes the present invention with way of example, and are not intended to limit the present invention's scope required for protection.
Make up HPV L1 mutain expression vector
Gene clone employing rite-directed mutagenesis PCR reacts and carries out; The original template that uses comprises pT0-T7-HPV16L1N30C plasmid (in table 1, being abbreviated as 16L1); PT0-T7-HPV52L1N40C plasmid (in table 1, being abbreviated as 52L1), pT0-T7-HPV6L1N5C plasmid (in table 1, being abbreviated as 6L1).Template, the primer of each PCR reaction are seen table 1, and PCR reaction amplification condition is made as: 94 ℃ of sex change 10 minutes, and 25 round-robin " 72 ℃ were extended 50 seconds for 94 ℃ of sex change 50 seconds, assigned temperature annealing certain hour ", last 72 ℃ were extended 10 minutes.The sequence of employed PCR primer is listed in table 2.
Table 1. makes up proteic each the PCR reaction conditions of sudden change HPV L1
Table 2: be used for making up the sudden change proteic primer sequence of HPV L1 (SEQ ID NO:19-30)
Pcr amplification obtains product (50 μ L) and adds 2 μ L DpnI restriction enzymes, handles 60min at 37 ℃.Get 10 μ L and transform competence intestinal bacteria ER2566 (available from New England Biolabs, Inc. (US) Massachusetts, United States of America) bacterium of 40 μ L with the Calcium Chloride Method preparation; It is coated contain kantlex (final concentration 25mg/mL; Solid LB substratum (LB medium component: 10g/L peptone, 5g/L yeast powder, 10g/L sodium-chlor down together); Down with), and 37 ℃ leave standstill cultivate 10-12 hour clear and legible to single bacterium colony.Picking list bacterium colony is to the test tube that contains 4mL liquid LB substratum (containing kantlex), 37 ℃ 220 rev/mins following shaking culture 10 hours, therefrom gets 1mL bacterium liquid in-70 ℃ of preservations.Utilize the T7 primer, record the segmental nucleotide sequence of the purpose of inserting in the pT0-T7 plasmid and be respectively SEQ ID NO:13,14,15,16,17,18, its amino acid sequence coded is SEQ ID NO:4,5,6,7,8,9.
The great expression of HPV L1 mutain
From-70 ℃, take out the Escherichia coli bacteria liquid that carries recombinant plasmid pT0-T7-HPV16L1-C175A, pT0-T7-HPV16L1-Δ C428, pT0-T7-HPV52L1-C175A, pT0-T7-HPV52L1-Δ C428, pT0-T7-HPV6L1-C175S, pT0-T7-HPV6L1-Δ C428; Inoculate respectively in the LB liquid nutrient medium that 100ml contains kantlex; At 200rpm, cultivated about 8 hours down for 37 ℃; Switching is gone into 15 bottles of 500ml and is contained in the LB substratum of kantlex (connecing 1ml bacterium liquid) respectively then.When bacterial concentration reaches OD600 and is 0.6 left and right sides, culture temperature is reduced to 25 ℃, every bottle added 500 μ L IPTG inducing culture 8 hours, centrifugal collection thalline.HPV16L1-C175A, HPV16L1-Δ C428, HPV52L1-C175A, HPV52L1-Δ C428, HPV6L1-C175S, the proteic thalline of HPV6L1-Δ C428 have been expressed in acquisition.
Express the bacterial cell disruption of HPV L1 mutain
Press the corresponding 10mL lysate of 1g thalline (20mM Tris damping fluid, pH7.2, the resuspended above-mentioned thalline that obtains of ratio 300mMNaCl).Adopt the broken thalline 30min of ultrasonoscope.With the centrifugal bacterial cell disruption liquid of 13500rpm (30000g) 15min, leave and take supernatant (that is broken bacterium supernatant).
The chromatogram purification of HPV L1 mutain
Instrument system: the AKTA explorer 100 type preparative liquid chromatography systems that GE Healthcare company (former Amershan Pharmacia company) produces.
Chromatography media: SP Sepharose 4 Fast Flow (GE Healthcare company), CHT-II (available from Bio-RAD), Butyl Sepharose 4 Fast Flow (GE Healthcare company).
Damping fluid: 20mM phosphate buffered saline buffer pH8.0,20mM DTT
20mM phosphate buffered saline buffer pH8.0,20mM DTT, 2M NaCl
Sample is the bacterial cell disruption supernatant of the HPV16L1-C175A that obtains of aforesaid method, HPV16L1-Δ C428, HPV52L1-C175A, HPV52L1-Δ C428, HPV6L1-C175S, HPV6L1-Δ C428
Elution program is: carry out cation exchange purification with SP Sepharose 4 Fast Flow earlier: 400mM NaCl wash-out foreign protein, and 800mM NaCl wash-out target protein is collected 800mM NaCl elutriated fraction; Then back 800mM NaCl elutriated fraction is carried out CHTII (hydroxylapatite chromatography) purifying: with the 800mM NaCl elutriated fraction of last step acquisition; With the NaCl concentration dilution to 0.5M; 500mM NaCl wash-out foreign protein; 1000mMNaCl wash-out target protein, the elutriated fraction when collecting 1000mM NaCl concentration.The 1000mM NaCl elutriated fraction that at last last step is obtained is carried out HIC (hydrophobic interaction chromatograph) purifying: 1000mM NaCl wash-out foreign protein, 200mM NaCl wash-out target protein, the elutriated fraction when collecting 200mM NaCl concentration.
The renaturation and the morphologic detection of embodiment 2:HPV L1 mutain
The renaturation of mutain
The purity of getting 2ml embodiment 1 gained is greater than 98% HPV16L1-C175A, HPV16L1-Δ C428, HPV52L1-C175A, HPV52L1-Δ C428, HPV6L1-C175S, HPV6L1-Δ C428 mutain or separately or in 2L renaturation buffer (50mM PB (sodium phosphate buffer) pH 6.0; 2mM CaCl2; 2mM MgCl2,0.5M NaCl) fully exchange of dialysis.After having exchanged, (20mM PB (sodium phosphate buffer) pH 6.5,0.5M NaCl) exchanges with 2L store buffer liquid.
The morphologic detection of HPV L1 mutain
HPV16L1-C175A, HPV16L1-Δ C428, HPV52L1-C175A, HPV52L1-Δ C428 albumen after renaturation is assembled are as stated above carried out transmission electron microscope observing.The 100kV transmission electron microscope that the instrument that uses is produced as company of NEC, magnification is 100,000 times.HPV16L1-C175A after the renaturation, HPV16L1-Δ C428, HPV52L1-C175A, HPV52L1-Δ C428 albumen with 2% phospho-wolframic acid pH7.0 negative staining, are fixed on the copper mesh of spray charcoal, observe.Electronic Speculum result sees Fig. 2-5, and wherein all the diameter of visible a large amount of homogeneous is the capsomere about 8nm.
Embodiment 3:HPV16 L1-C175A learns with different assembling damping fluid assembling heterozygosis VLP and particle form with HPV52L1-Δ C428 and detects
HPV16 L1-C175A assembles heterozygosis with HPV52L1-Δ C428 with different assembling damping fluids
VLP
Measure the protein concentration of HPV16L1-C175A, HPV52L1-Δ C428; (about 2ml) HPV16L1-C175A and the HPV52L1-Δ C428 that gets certain volume dialyses in 2L store buffer liquid (20mM PB (sodium phosphate buffer) pH6.5,0.5M NaCl) after according to 1: 1 mixed of mass ratio; 2L renaturation buffer (50mM PB (sodium phosphate buffer) pH6.0,2mM CaCl
2, 2mM MgCl
2, 0.5M NaCl); 20mM PB (sodium phosphate buffer) pH 7.0 and 0.5M NaCl; Exchange 12h in three kinds of damping fluids.
Hybrid virus appearance particle form is learned and is detected
Sample under above-mentioned three kinds of assembling conditions is got 100 μ L carry out transmission electron microscope observing.The 100kV transmission electron microscope that the instrument that uses is produced as company of NEC, magnification is 100,000 times.Sample under four kinds of assembling conditions is got 13.5 μ L with 2% phospho-wolframic acid pH7.0 negative staining, be fixed on the copper mesh of spray charcoal, observe.Electronic Speculum result sees Fig. 3; Wherein the visible a large amount of radiuses of Fig. 3 A and 3B are the virus-like particle about 25nm; Further relatively can find the particle size homogeneous comparatively among Fig. 3 B, and the visible a large amount of pentamers of radius about 5nm of Fig. 3 C there is not virus-like particle to exist.This shows that HPV16 L1-C175A and HPV52L1-Δ C428 can be at store buffer liquid (20mM PB (sodium phosphate buffer) pH 6.5,0.5M NaCl), renaturation buffer (50mM PB (sodium phosphate buffer) pH 6.0,2mM CaCl
2, 2mM MgCl
2, 0.5M NaCl) assemble under the condition, can not under 20mM PB (sodium phosphate buffer) pH 7.0 and 0.5MNaCl condition, assemble, at renaturation buffer (50mM PB (sodium phosphate buffer) pH 6.0,2mM CaCl
2, 2mM MgCl
2, 0.5M NaCl) and the particle size of assembling under 0.5MNaCl condition homogeneous comparatively.
Embodiment 4:HPV16 L1-C175A assembles heterozygosis VLP and graininess analysis with HPV52L1-Δ C428 with different assembling ratios
The different assembling ratio assembling of HPV16 L1-C175A heterozygosis with HPV52L1-Δ C428
VLP
Measure the protein concentration of HPV16L1-C175A, HPV52L1-Δ C428, (about 2ml) HPV16L1-C175A and the HPV52L1-Δ C428 that gets certain volume dialyses after according to 2.5: 1,2: 1,1: 1,1: 2 mixed of mass ratio and in the 2L renaturation buffer, exchanges 12h.
Sieve chromatography is analyzed hybrid virus appearance graininess
Adopt 1120 Compact LC highly effective liquid phase chromatographic systems of U.S. Agilent company to carry out chromatography, the analytical column that uses is TSK Gel PW5000xl 7.8x300mm.PBS pre-balance chromatography column to the absorption value at 280nm place with 2 times of column volumes does not have obvious variation, and the absorption value of detector is made zero.By automatic sampler sample introduction and analytical results.The result is as shown in Figure 4; The protein peak that the albumen that HPV16L1-C175A and HPV52L1-Δ C428 assemble in varing proportions occurs at first is all about 12min; Suitable with HPV16VLP, than HPV52L1-Δ C428 RT in advance, this shows with the assembling of aforementioned proportion heterozygosis all can form particle form; Second protein peak of above-mentioned albumen is about 16min; Suitable with HPV52L1-Δ C428 RT; Explanation is remaining pentamer for assembling, further relatively can find, and is minimum in second protein peak peak height of heterozygosis VLP residue of mass ratio ratio assembling in 1: 1; Remaining pentamer is minimum when showing its assembling, and the assembling ratio is the most suitable.
Hybrid virus appearance particle form is learned and is detected
Sample under above-mentioned three kinds of assembling conditions is got 100 μ L carry out transmission electron microscope observing.The 100kV transmission electron microscope that the instrument that uses is produced as company of NEC, magnification is 100,000 times.Sample under four kinds of assembling conditions is got 13.5 μ L with 2% phospho-wolframic acid pH7.0 negative staining, be fixed on the copper mesh of spray charcoal, observe.Electronic Speculum result sees Fig. 6-8.Observations is seen Fig. 5, wherein all visible a large amount of virus-like particles; Further relatively can find that Fig. 5 C is big or small homogeneous in the visual field, the virus-like particle about radius 30nm shows the particle size homogeneous that HPV16 L1-C175A and HPV52L1-Δ C428 form according to mass ratio assembling in 1: 1.
The three-dimensional structure of embodiment 5:HPV16 L1-C175A and HPV52L1-Δ C428 virus-like particle is rebuild
Use freezing Electronic Speculum three-dimensional structure to rebuild experiment (Wolf M; Garcea RL; Grigorieff N.et al.Proc Natl Acad Sci U S A. (2010), 107 (14): 6298-303.) rebuild HPV16 L1-C175A and HPV52L1-Δ C428 hybrid virus appearance particulate three-dimensional structure.In brief; (Fig. 6 A) chooses 372 big or small homogeneous respectively in the freezing sem image of HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP, diameter carries out the overlapping and structural remodeling of computingmachine above the particle of 50nm, thereby obtains the three-dimensional structure of HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis.The three-dimensional structure that is obtained is shown in Fig. 6 B, and wherein the resolving power of HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP is
.The result shows, HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP be by the icosahedral structure of virus of the T=7 of 72 capsomeres (form subunit, pentamer) formation (h=1, k=2).Different with the general icosahedron viruses capsid that meets the quasi-equivalence principle, all composition subunits are pentamer in HPV16 L1-C175A and the HPV52L1-Δ C428 heterozygosis VLP structure, and do not see six aggressiveness, and the outermost diameter of VLP is about 60nm.This three-dimensional structure with the HPV VLP in natural HPV virion of reporting before and eukaryotic expression system (for example, pox viruses express system) source is similar.
Embodiment 6:HPV16 L1-C175A and HPV52L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428 heterozygosis VLP checking
The SDS-polyacrylamide gel electrophoresis detects
Get the HPV16 L1-C175A and the HPV52L1-Δ C428 heterozygosis VLP sample 100 μ L of embodiment 5 purifying, add 20 μ L 6X Loading Buffer, mixing and in 80 ℃ of water-bath 10min; Get then 10 μ l in the 10%SDS-polyacrylamide gel with 120V voltage electrophoresis 120min; Show electrophoretic band with Coomassie brilliant blue dyeing then.Electrophoresis result is seen Fig. 7.Electrophoresis result shows that HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP have two specific bands, and consistent with the band of HPV16L1, HPV52L1 respectively, the two mass ratio is about 1: 1.
Protein immunoblotting detects (western-blot)
HPV16 L1-C175A and HPV52L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428 heterozygosis VLP to embodiment 5 purifying carries out electrophoresis as stated above; After electrophoresis finishes; Use HPV16 specific antibody 21A5 and HPV52 specific antibody 12D10 to carry out Western respectively and detect, the result sees Fig. 8-9.The result shows that the special linear antibody 12D10Western reaction of HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP and HPV52 has specific band, and the two mass ratio is about 1: 1.
Embodiment 7:HPV L1 mutain, HPV VLP, HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP thermostability are estimated
Use is estimated HPV L1 mutain, HPV VLP, HPV16 L1-C175A and HPV52L1-Δ C428 heterozygosis VLP thermostability available from the differential temperature calorimeter VP Capillary DSC of U.S. GE company (former MicroCal company).Use proteic store buffer liquid as contrast, with the temperature rise rate of 90 ℃/min, each albumen is scanned in 10 ℃ of-90 ℃ of intervals, detected result is seen Figure 10.The result shows that the melting temperature (Tm) of HPV16 L1-C175S and HPV52L1-Δ C428 heterozygosis VLP is 67.63 ℃; 66.98 ℃ of melting temperature (Tm)s that are higher than mutain HPV16 L1-C175S and HPV52L1-Δ C428 with 56.86 ℃; Between 69.15 ℃ and 64.41 ℃ of the melting temperature (Tm)s of HPV16 VLP and HPV52 VLP; Explain that HPV16 L1-C175S and HPV52L1-Δ C428, HPV52L1-C175S and HPV16L1-Δ C428 heterozygosis VLP thermostability are better than pentamer, suitable with the stability of VLP.
Embodiment 8:HPV16 L1-C175A and HPV52L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428 heterozygosis VLP immune protective are estimated
Use mouse to estimate the immune protective of HPV16 L1-C175A of the present invention and HPV52L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428 heterozygosis VLP.It is 4 all regular grade mouse in age (available from Shanghai Si Laikang laboratory animal ltd) that animal is used in immunity.Heterozygosis particle that embodiment 2 is prepared and HPV16VLP and HPV52VLP are adsorbed on the aluminum hydroxide adjuvant.Be divided into 5 groups by different immunizing antigens, 3 mouse of every group of immunity.Immune programme for children is: initial immunity during 0 week; Each is strengthened once during 2 and 4 weeks.Immunization ways is an abdominal injection, carries out immunity according to immunizing antigen shown in the table 2 and dosage.Behind the initial immunity, the 8th week was extracted eyeball venous blood, and separation of serum detects NAT.Detected result is shown in figure 12.The result shows; The HPV16 L1-C175A and HPV52L1-Δ C428, HPV52L1-C175A and the HPV16L1-Δ C428 heterozygosis VLP that obtain according to the said method of embodiment 1-4 have good immunogenicity; Can in the mouse body, induce the neutralizing antibody to HPV16, HPV52 of high titre; The effective vaccine that can be used as prevention HPV16, HPV52 infection; In addition, HPV16L1-C175A and HPV52L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428 heterozygosis VLP also induce and have produced certain neutralizing antibody to HPV58.Except using freund's adjuvant, this vaccine also can use other adjuvants well known in the art, for example white lake or phosphagel phosphaljel adjuvant.
Table 3 immunizing dose and programsheet
Embodiment 9:HPV16L1-C175A and HPV6L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428, HPV52L1-C175A and HPV6L1-Δ C428, HPV6L1-C175S and HPV16L1-Δ C428, HPV6L1-C175S and HPV52L1-Δ C428 hybrid virus appearance granules prepn and morphological observation
The assembling of hybrid virus appearance particulate
The purity of getting 2ml embodiment 1 gained greater than 98% heterozygote albumen by with HPV16L1-C175A and HPV6L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428, HPV52L1-C175A and HPV6L1-Δ C428, HPV6L1-C175S and HPV16L1-Δ C428, HPV6L1-C175S and HPV52L1-Δ C428 combination by 1: 1 proportioning in fully exchange of 2L renaturation buffer dialysis; After having exchanged, exchange with 2L store buffer liquid.
Hybrid virus appearance particle form is learned and is detected
The sample of assembling under above-mentioned five kinds of combination conditions is got 100 μ L carry out transmission electron microscope observing.The 100kV transmission electron microscope that the instrument that uses is produced as company of NEC, magnification is 100,000 times.Sample under four kinds of assembling conditions is got 13.5 μ L with 2% phospho-wolframic acid pH, 7.0 negative staining, be fixed on the copper mesh of spray charcoal, observe.Electronic Speculum result sees Figure 12.Figure 12 shows that these albumen can be assembled and form a large amount of radiuses is the virus-like particle about 25nm, and particle size conforms to theoretical size, and uniformity.
In addition; Through the method susceptible of proof that uses embodiment 8 to describe; HPV16L1-C175A that the present invention obtained and HPV6L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428, HPV52L1-C175A and HPV6L1-Δ C428, HPV6L1-C175S and HPV16L1-Δ C428, HPV6L1-C175S and HPV52L1-Δ C428 heterozygote VLP have good immunogenicity equally; Can induce the neutralizing antibody of high titre in animal body, thereby can be used as the vaccine that prevention HPV infects.
Although embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that according to disclosed all instructions can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by accompanying claims and any equivalent thereof.
Claims (8)
1. comprise the proteic hybrid virus appearance of other HPV L1 of two or more different shaped particle, displacement and/or disappearance take place at the 175th with 428 halfcystines in wherein said HPV L1 albumen.
2. the hybrid virus appearance particle of claim 1, wherein said hybrid virus appearance particle comprise and derive from a kind of genotypic HPV L1-C175 replacement mutation body and from the HPV L1-C428 deletion mutant of another kind of different genotype.
3. the hybrid virus appearance particle of claim 1, wherein said hybrid virus appearance particle comprise and derive from a kind of genotypic HPV L1-C428 replacement mutation body and from the HPV L1-C175 deletion mutant of another kind of different genotype.
4. each hybrid virus appearance particle of claim 1-3, the replacement mutation body of wherein said HPV L1 is selected from: C175A replacement mutation body, C175S replacement mutation body, C428A replacement mutation body, C428S replacement mutation body.
5. each hybrid virus appearance particle of claim 1-3, the deletion mutant of wherein said HPV L1 is selected from: Δ C175 deletion mutant, Δ C428 deletion mutant.
6. each hybrid virus appearance particle of claim 1-5; Wherein said HPV L1 protein mutant is derived from following two kinds or more kinds of HPV genotype: HPV 6,11,16,18,31,33,45,52,58 and 59; Be preferably HPV 16,52 and 6, most preferably be HPV16 L1-C175A and HPV52L1-Δ C428, HPV52L1-C175A and HPV16L1-Δ C428, HPV16L1-C175A and HPV6L1-Δ C428, HPV52L1-C175A and HPV6L1-Δ C428, HPV6L1-C175S and HPV16L1-Δ C428 or HPV6L1-C175S and HPV52L1-Δ C428 hybrid virus appearance particle.
7. each hybrid virus appearance particle of claim 1-6, wherein being assembled into the proteic proportioning of other HPV L1 of two kinds of different shaped of hybrid virus appearance particulate is 1: 1,2: 1 or 1: 2.
8. each hybrid virus appearance particulate method of preparation claim 1-7 comprises:
A) at said sudden change of expression in escherichia coli or disappearance HPV L1 albumen,
B) will express said sudden change or disappearance HPV L1 protein purification, obtain the sudden change or the disappearance HPV L1 albumen of purity more than 95%,
C) with b) sudden change of purifying or the HPV L1 albumen of disappearance were according to 1: 1 mixed in molar ratio, and reductive agent is removed in dialysis, promptly obtains heterozygosis VLP;
Preferably, said method comprises prepared hybrid virus appearance particle and pharmaceutically acceptable carrier and/or mixed with excipients, is used to prevent HPV to infect or infects the disease that caused such as the vaccine of cervical cancer by HPV with preparation.
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