CN104418942A - Truncated L1 proteins of human papilloma virus (HPV), virus-like particles as well as preparation method and application of virus-like particles - Google Patents

Truncated L1 proteins of human papilloma virus (HPV), virus-like particles as well as preparation method and application of virus-like particles Download PDF

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CN104418942A
CN104418942A CN201310388695.0A CN201310388695A CN104418942A CN 104418942 A CN104418942 A CN 104418942A CN 201310388695 A CN201310388695 A CN 201310388695A CN 104418942 A CN104418942 A CN 104418942A
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albumen
seq
viruslike particle
hpv
sequence
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孔维
姜春来
孙博
徐菲
宋海鹏
张喆
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CHANGCHUN BCHT BIOTECHNOLOGY Co Ltd
Jilin University
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CHANGCHUN BCHT BIOTECHNOLOGY Co Ltd
Jilin University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention relates to truncated L1 proteins of a human papilloma virus (HPV), virus-like particles as well as a preparation method and application of virus-like particles. The invention particularly relates to HPV6-type, HPV11-type, HPV16-type, HPV18-type or HPV58-type truncated L1 proteins of the HPV, the virus-like particles as well as the preparation method and application of the virus-like particles.

Description

The L1 albumen, its viruslike particle and its preparation method and application of the human papillomavirus of brachymemma
Technical field
The present invention relates to the L1 albumen of the human papillomavirus of brachymemma, its viruslike particle, with and its production and use.Particularly, the present invention relates to the truncation type L1 albumen of human papillomavirus HPV6 type, HPV11 type, HPV16 type, HPV18 type or HPV58 type, its viruslike particle, with and its production and use.
Background technology
Human papillomavirus (Human Papillomavirus, HPV) is a class double-chain small molecule DNA virus, has strict species specificity, the skin of main infection people and mucosal tissue, causes the proliferative lesion of corresponding site epithelium.According to nucleic acid sequence homology, HPV can be divided into 100 many types of.HPV16,18 and 58 and the generation height correlation of cervical cancer, wherein HPV16 is topmost carcinogenic factor.HPV6 and 11 is more than 90% Genital warts and the papillomatous virulence factor of larynx.
Complete human papillomavirus particle is made up of virus genom DNA and capsid two portions.Viral genome is double-stranded cyclic DNA (about 8kb), is divided into three functional zone according to function: early stage district, 6 Nonstructural Proteins (E1, E2, E4, E5, E6, E7) of encode, and described albumen copies with viral, it is relevant to transcribe with transformation; Late region, containing L1 and L2 two ORF, coding Major capsid protein L1 and secondary capsid protein L2 two structural protein, participate in the assembling of virus particle; LCR (LCR), also known as non-coding region or upstream regulatory region, any albumen of not encoding, but containing multiple controlling elements such as replication orgin, promotor, enhanser, silencers, viral the copying and transcribe of impact.
HPV L1 albumen is human papillomavirus major capsid protein, and molecular weight is 55-60kDa, is HPV vaccine major structural protein.The HPV L1 albumen of expressing in multiple expression system is assisted without the need to L2 albumen, can be formed in viruslike particle (Virus-Like Particle, VLP) like morphological structure and natural viral Particle Phase.This virus-like particle remains the natural epitopes of virion, has stronger immunogenicity, and can induce in homotype HPV virus and the body of Hangzhoupro, be HPV preventative vaccine main direction of studying.In experimentation on animals, the antibody that the VLP only having L1 albumen to be assembled into brings out has the ability of neutralization virus, preventing infection, and can not preventing infection after the L1 protein immunization of sex change.
The main viruslike particle adopting prokaryotic system intestinal bacteria and eukaryotic system yeast saccharomyces cerevisiae to express HPV in prior art.But after the HPV L1 protein translation expressed by prokaryotic system, degree of modification is low, correctly cannot fold voluntarily, mostly lose its native conformation, mainly exists with occlusion body form.Although by occlusion body purifying, the steps such as renaturation also can obtain the VLP of HPV, in renaturation process, loss of proteins amount is large, low by yield, is difficult to apply in large-scale industrial production.Yeast saccharomyces cerevisiae also has its limitation as a kind of host of expressing heterologous albumen.When expanding to technical scale by laboratory, its output declines rapidly.Reason is that the selective pressure maintaining plasmid high copy number in substratum disappears, and plasmid becomes unstable, and copy number declines.Copy number is the necessary factors of high expression, and therefore copy number declines, and also directly causes the decline of exogenous gene expression amount.The protein of yeast saccharomyces cerevisiae secretion also may be modified by excessive glycosylation, and the glycosylation modified meeting of this mistake seriously changes the immunogenicity of protein, reduces active, shortens the time of being detained in blood.The numerous protein of yeast saccharomyces cerevisiae secretion, is not secreted in substratum, but to be trapped in periplasmic space with the mode of Cell binding, has manufactured difficulty to protein purification in scale operation.
The advantage of baculovirus-insect cell system is that security is high, large to exogenous gene cloning capacity, there is the high expression under polyhedron promoter control, post translational processing is modified, without features such as contaminated with endotoxins, but in large-scale industrial production, there is the problem that expression amount is low, toxigenic capacity is high, make the with high costs of the finished product, limit its application.Therefore still need in this area to improve the expression amount of HPV L1 albumen in baculovirus-insect cell system, and in body, bring out the neutralizing antibody of high titre, reduce the cost of scale operation HPV vaccine further.
Summary of the invention
The present inventor finds, compared with wild-type HPV L1 gene, codon through optimize and remove C and hold the expression amount of HPV L1 gene in baculovirus-insect cell system of nuclear localization signal to be significantly improved; Find simultaneously compared with wild-type HPV L1 viruslike particle, remove after C holds nuclear localization signal HPV L1 viruslike particle immune animal and can induce the higher specificity neutralizing antibody for HPV, complete the present invention on this basis.
The invention provides a kind of C that removes and hold the HPV L1 albumen of nuclear localization signal, consisting of viruslike particle and vaccine containing this viruslike particle.
An aspect of of the present present invention relates to HPV L1 gene, comprises the HPV L1 gene be optimized according to insect cell preference codon.
One aspect of the present invention relates to HPV, in particular to HPV6 type, HPV11 type, HPV16 type, HPV18 type or HPV58 type.
One aspect of the present invention relates to following HPV L1 albumen:
C holds brachymemma 9 or 21 amino acid whose HPV6 type L1 albumen; Preferably, the sequence of this truncated protein is SEQ ID NO:1 or 2; More preferably, the sequence of this truncated protein is SEQ ID NO:2;
C holds brachymemma 9 or 21 amino acid HPV11 type L1 albumen; Preferably, the sequence of this truncated protein is SEQ ID NO:3 or 4; More preferably, the sequence of this truncated protein is SEQ ID NO:4;
C holds brachymemma 7 or 22 amino acid whose HPV16L1 albumen; Preferably, the sequence of this truncated protein is SEQ ID NO:5 or 6; More preferably, the sequence of this truncated protein is SEQ ID NO:6;
C holds brachymemma 9 or 24 amino acid whose HPV18L1 albumen; Preferably, the sequence of this truncated protein is SEQ ID NO:7 or 8; More preferably, the sequence of this truncated protein is SEQ ID NO:8; Or
C holds brachymemma 7 or 19 amino acid whose humanpapilloma virus 58 L1 proteins; Preferably, the sequence of this truncated protein is SEQ ID NO:9 or 10; More preferably, the sequence of this truncated protein is SEQ ID NO:10.
One aspect of the present invention relates to coding C and holds the HPV L1 albumen of brachymemma and the HPV L1 gene of codon through optimizing, and is optimized according to insect cell preference codon, wherein:
Described C holds the HPV L1 albumen of brachymemma to be that C holds brachymemma 9 or 21 amino acid HPV6 type L1 albumen; Preferably, the sequence of described HPV L1 gene is SEQ ID NO:11 or 12; More preferably, the sequence of described HPV L1 gene is SEQ ID NO:12;
Described C holds the HPV L1 albumen of brachymemma to be that C holds brachymemma 9 or 21 amino acid HPV11 type L1 albumen; Preferably, the sequence of described HPV L1 gene is SEQ ID NO:13 or 14; More preferably, the sequence of described HPV L1 gene is SEQ ID NO:14;
Described C holds the HPV L1 albumen of brachymemma to be that C holds brachymemma 7 or 22 amino acid whose HPV16L1 albumen; Preferably, the sequence of described HPV L1 gene is SEQ ID NO:15 or 16; More preferably, the sequence of described HPV L1 gene is SEQ ID NO:16;
Described C holds the HPV L1 albumen of brachymemma to be that C holds brachymemma 9 or 24 amino acid whose HPV18L1 albumen; Preferably, the sequence of described HPV L1 gene is SEQ ID NO:17 or 18; More preferably, the sequence of described HPV L1 gene is SEQ ID NO:18; Or
Described C holds the HPV L1 albumen of brachymemma to be that C holds brachymemma 7 or 19 amino acid whose humanpapilloma virus 58 L1 proteins; Preferably, the sequence of described HPV L1 gene is SEQ ID NO:19 or 20; More preferably, the sequence of described HPV L1 gene is SEQ ID NO:20.
And the carrier containing said gene.
One aspect of the present invention relates to the cell comprising above-mentioned carrier, and described cell comprises the insect cells such as sf9, sf21, HighFive.
One aspect of the present invention relates to and comprises the above-mentioned C end L1 albumen of brachymemma or the composition of its polynucleotide or carrier or cell.
One aspect of the present invention relates to HPV6 type, HPV11 type, HPV16 type, HPV18 type or HPV58 type viruslike particle, and wherein above-mentioned viruslike particle comprises:
C holds brachymemma 9 or 21 amino acid HPV6 type L1 albumen; Preferably,
Described C holds the sequence of the HPV L1 albumen of brachymemma to be SEQ ID NO:1 or 2;
More preferably, C holds the sequence of the HPV L1 albumen of brachymemma to be SEQ ID NO:2;
C holds brachymemma 9,21 amino acid HPV11 type L1 albumen; Preferably,
Described C holds the sequence SEQ ID NO:3 or 4 of the HPV L1 albumen of brachymemma;
More preferably, C holds the sequence of the HPV L1 albumen of brachymemma to be SEQ ID NO:4;
C holds brachymemma 7,22 amino acid HPV16 type L1 albumen; Preferably,
Described C holds the sequence SEQ ID NO:5 or 6 of the HPV L1 albumen of brachymemma;
More preferably, C holds the sequence of the HPV L1 albumen of brachymemma to be SEQ ID NO:6;
C holds brachymemma 9,24 amino acid HPV18 type L1 albumen; Preferably,
Described C holds the sequence SEQ ID NO:7 or 8 of the HPV L1 albumen of brachymemma;
More preferably, C holds the sequence of the HPV L1 albumen of brachymemma to be SEQ ID NO:8; Or
C holds brachymemma 7,19 amino acid HPV58 type L1 albumen; Preferably,
Described C holds the sequence SEQ ID NO:9 or 10 of the HPV L1 albumen of brachymemma;
More preferably, C holds the sequence of the HPV L1 albumen of brachymemma to be SEQ ID NO:10; .
Further aspect of the present invention relates to a kind of method obtaining HPV6 type, HPV11 type, HPV16 type, HPV18 type or HPV58 type viruslike particle, it is included in the HPV6 type of the aforementioned brachymemma of baculovirus-insect cells, HPV11 type, HPV16 type, HPV18 type or HPV58 type L1 gene fragment, cell described in cracking, then carries out purification process by the cracking supernatant of the HPV L1 albumen containing above-mentioned brachymemma.
The invention still further relates to preventative HPV vaccine, it comprises the viruslike particle formed by HPV16L1 albumen of the present invention.Preferably, this vaccine also comprises at least one and is selected from following viruslike particle: HPV6 viruslike particle, the HPV11 viruslike particle formed by HPV11L1 albumen, the HPV18 viruslike particle formed by HPV18L1 albumen and the HPV58 viruslike particle formed by humanpapilloma virus 58 L1 protein formed by HPV6L1 albumen of the present invention.
In one embodiment, described vaccine contains the HPV58 viruslike particle formed by humanpapilloma virus 58 L1 protein of the present invention.Preferably, described vaccine contains the HPV58 viruslike particle formed by the HPV58 type L1 albumen with SEQ ID NO:10 protein sequence.More preferably, described vaccine is also containing the HPV16 viruslike particle formed by HPV16L1 albumen of the present invention and the HPV18 viruslike particle formed by HPV18L1 albumen, particularly containing the HPV16 viruslike particle formed by the HPV16 type L1 albumen with SEQ ID NO:6 protein sequence, containing the HPV18 viruslike particle formed by the HPV18 type L1 albumen with SEQ ID NO:8 protein sequence.
In one embodiment, described vaccine contains the HPV6 viruslike particle formed by HPV6L1 albumen of the present invention and the HPV11 viruslike particle formed by HPV11L1 albumen.Preferably, described vaccine contains the HPV6 viruslike particle that formed by the HPV6 type L1 albumen with SEQ ID NO:2 protein sequence and containing the HPV11 viruslike particle formed by the HPV11 type L1 albumen with SEQ ID NO:4 protein sequence.More preferably, described vaccine is also containing the HPV16 viruslike particle formed by HPV16L1 albumen of the present invention and the HPV18 viruslike particle formed by HPV18L1 albumen, particularly containing the HPV16 viruslike particle formed by the HPV16 type L1 albumen with SEQ ID NO:6 protein sequence, containing the HPV18 viruslike particle formed by the HPV18 type L1 albumen with SEQ ID NO:8 protein sequence.
In one embodiment, described vaccine contains HPV6 viruslike particle of the present invention, HPV11 viruslike particle and HPV58 viruslike particle.Preferably, described vaccine contain formed by the HPV6 type L1 albumen with SEQ ID NO:2 protein sequence HPV6 viruslike particle, containing the HPV11 viruslike particle that formed by the HPV11 type L1 albumen with SEQ ID NO:4 protein sequence with contain the HPV58 viruslike particle formed by the HPV58 type L1 albumen with SEQ ID NO:10 protein sequence.More preferably, described vaccine is also containing the HPV16 viruslike particle formed by HPV16L1 albumen of the present invention and the HPV18 viruslike particle formed by HPV18L1 albumen, particularly containing the HPV16 viruslike particle formed by the HPV16 type L1 albumen with SEQ IDNO:6 protein sequence, containing the HPV18 viruslike particle formed by the HPV18 type L1 albumen with SEQ ID NO:8 protein sequence.
Compared to prior art, the present invention selects codon optimized design and C holds the HPV6L1 gene of brachymemma, HPV11L1 gene, HPV16L1 gene, HPV18L1 gene or HPV58L1 gene, be expressed in shape virus-insect cell expressioning system, comparatively wild-type HPV6 type, HPV11 type, HPV16 type, HPV18 type or HPV58 type L1 expressing quantity are significantly improved to make the HPV L1 expressing quantity of above-mentioned brachymemma.Under Electronic Speculum, 50-60nm is viewed as after expression gained viruslike particle is purified, similar to wild-type HPV L1 virion.With the HPV6 type of the C of codon optimized design end brachymemma, HPV11 type, HPV16 type, HPV18 type or HPV58 type viruslike particle immune mouse, comparatively wild-type HPV6 type, HPV11 type, HPV16 type, HPV18 type or HPV58 type, can produce the HPV specificity neutralizing antibody of more high-titer.
Experiment proves: the VLP of the HPV total length L1 albumen of Sf9 insect cell expression is mainly positioned Sf9 nucleus.Do not affect the formation of VLP after C-end brachymemma nuclear localization signal, and its VLP formation efficiency improves greatly.Therefore, not affecting in VLP formation and bioactive situation, making expressed L1 albumen mainly be positioned endochylema, and not entering nucleus, thus making L1 protein purification become relatively easy, and can also expression level be increased.
Removing C holds truncation type HPV16L1 albumen (HPV16-483) NAT of nuclear localization signal to be significantly higher than full-length (HPV16-505), illustrates that the humoral immunoresponse(HI) level that truncation type brings out is significantly higher than full-length.This may be because the HPV16VLP of removal nuclear localization signal is trapped in tenuigenin, and the HPV16VLP containing nuclear localization signal is positioned in nucleus, and the truncation type HPV16VLP in tenuigenin is easier in passing bone-marrow-derived lymphocyte than total length HPV16VLP, bring out specific humoral immunoresponse(HI).Known by above-mentioned result of study, the HPV VLP removing nuclear localization signal is easier to by bone-marrow-derived lymphocyte identification, is conducive to the neutralizing antibody producing high titre.
Embodiment
Below in conjunction with embodiment, the present invention is illustrated description further.
Embodiment 1 codon optimized design and synthesis human mammilla tumor virus L 1 gene
According to the principle such as codon-bias, mRNA secondary structure, mRNA free energy stability, GC content that insect cell uses, design total length HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV58 type nucleotide sequence (the corresponding sequence see in sequence table).By JaRa company respectively business synthesize above-mentioned full-length gene.
The various HPV L1 albumen of embodiment 2-restructuring rod granule builds
Table 1. primer table:
Primer Sequence
6F GGATCCGCCACCATGTGGCG
6R500 CTCGAGTCAGCGCTTGGTC
6R491 CTCGAGTCAGGGTGCGGCGC
6R479 CTCGAGTCAGACGCCTGTGC
11F GGATCCATGTGGCGCCCAT
11R501 CTCGAGTCACTTTTTGGTC
11R492 CTCGAGTCAGGCGGTGCTGGG
11R480 CTCGAGTCATCTCTTGATGCCG
16F GCGGCCGCGCCACCATGAGCCTGT
16R505 CTCGAGTCACAGCTTCCTCTTCTTC
16R498 CTCGAGTCAGGCGGTGGTGCTGG
16R483 CTCGAGTCACAAGTTCACCCTGGGC
18F GGATCCGCCACCATGAGCGT
18R507 CTCGAGTCACTTCTTCACCTTCTTCC
18R498 CTCGAGTCAGCTGCTGGTGGTTGCGC
18R483 CTCGAGTCACAGCCTGGGCTTGGC
58F GGATCCGCCACCATGGCC
58R498 CTCGAGTCACTTCTTCACCTTCTTCC
58R491 CTCGAGTCAGGTGCTGGGGGC
58R479 CTCGAGTCACAGGCCGCTCTG
The list of table 2. total length and truncation type HPV L1 and the primer thereof
With each hypotype HPV DNA genome (synthesis of JaRa company), for template, amplify object fragment with corresponding primer through PCR method, size is at about 1500bp respectively.Each object fragment is connected on T easy carrier.After order-checking is correct, then be connected with pFastBac-1 plasmid, obtain 10 kinds of transfer vectors containing goal gene respectively, cut qualification correctly through enzyme.
Reaction system:
Reaction conditions: in 0.2ml Eppendorf tube, 94 DEG C of denaturations 3 minutes; Enter circulation 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 2 minutes, circulate 25 times; 72 DEG C fully extend 7 minutes, and amplification obtains the DNA fragmentation of about 1.5kb size.This PCR primer is connected with pGEM-T easy carrier (purchased from promega), cuts qualification through BamHI/XhoI enzyme, obtain the positive colony containing the recombinant vectors pGEM T easy-HPV L1 inserting brachymemma HPV L1 gene.Two for each hypotype pGEM T easy-HPV L1 enzyme cutting is reclaimed each hypotype L1 segment respectively afterwards, connects in the pFast Bac-1 of same double digestion, obtain pFastBacl-HPV L1 recombinant plasmid.Ligation method: HPV L1DNA8 μ L, pFast Bac-1(is purchased from Invitrogen) 1 μ L, T4 ligase enzyme damping fluid 10 μ L, T4 ligase enzyme 1 μ L, 4 DEG C of connections are spent the night.Product conversion will be connected in DH5 α, extract plasmid in a small amount.Double digestion screens the recombinant plasmid of insertion in the right direction.
By each hypotype pFastBacl-HPV L1 Plastid transformation of building to competence Host Strains DH10BAC(purchased from Invitrogen) in, cultivate screening, make the Bacmid in donor plasmid pFastBac1l-HPV L1 and DH10BAC that swivel base restructuring occur, obtain the recombinant baculovirus shuttle vectors containing goal gene.
Cultivate the Host Strains transformed, extract the Bacmid of restructuring.Transformation of E. coli (DH10BAC), picking list bacterial plaque.The Bacmid(4 kind resistant gene of screening restructuring adds the screening of blue hickie), cultivate 36-48h, hickie is chosen in 4 DEG C of colour developings of spending the night.Used medium contains: penbritin (Amp) final concentration 100 μ g/mL, gentamicin (gentamicin, Gm) final concentration 7 μ g/mL, kantlex (kanamycin, Kan) final concentration 50 μ g/mL, tsiklomitsin (tetracycline, Tet) final concentration 10 μ g/mL; IPTG final concentration 40 μ g/mL, X-Gal final concentration 300 μ g/mL.
Bacmid extraction alkaline lysis method of extracting (adopt Tiangen company without the large extraction reagent kit of intracellular toxin plasmid (DP117)):
1) get bacterium liquid that 1.5ml grows to plateau to be added to 1.5ml EP and to manage, the centrifugal 1min of 12000g, supernatant discarded.
2) in precipitation, 300 μ L Solution1(TE solution are added, 50MmTris10mMEDTA) resuspended, then add 300 μ L Solution2 (1%SDS, 0.2MNaOH), mix gently, room temperature leaves standstill 5min.
3) 300 μ L Solution3(3MKAc, pH5.5 are slowly added), mix gently, ice bath 10min.
4) the centrifugal 10min of 12000g.
5) draw supernatant to new EP pipe, then add equal-volume Virahol, put upside down mixing, ice bath 10min.
6) room temperature, the centrifugal 15min of 12000g, inhales and abandons supernatant.
7) in precipitation, add 70% ethanol 500 μ L, put upside down for several times, the centrifugal 5min of 12000g,
Abandon supernatant, dry air.
8) by the 7th) step repeats once.
9) precipitation TE(pH8.0) to dissolve, 4 DEG C of preservations, long-term preservation puts-20 DEG C.
10) PCR checking is carried out extracting the rod granule obtained by M13 primer:
M13F CGCCAGGGTTTTCCCAGTCACGAC
M13R CACACAGGAAACAGCTATGAC。
Result shows, and obtains various HPV L1 albumen-restructuring rod granule.
Embodiment 3 is recombinated rod granule transfection and virus amplification
1. restructuring rod granule transfection
1) sf9 insect cell is cultivated: in 6 orifice plates of diameter 35mm, add 9X10 5individual cells/well, cell is in logarithmic phase, the survival rate of 97%; Sucking-off Grace substratum (purchased from Invitrogen), adds the Grace substratum of antibiotic-free serum-free, allows cell attachment at least 1h (27 DEG C or room temperature);
2) prep solution A liquid: draw the Grace substratum that 5 μ l restructuring Bacmid plasmid DNA are added to 100 μ l antibiotic-free serum-frees;
3) prep solution B liquid: by 6 μ l Cell FECTIN(purchased from Invitrogen) be added to the Grace substratum of 100 μ l antibiotic-free serum-frees, A liquid is mixed with B liquid, mixes gently, incubated at room 30min;
4) attached cell is washed twice, sucking liquid with the Grace substratum of antibiotic-free serum-free again, add 800 μ l Grace substratum in A, B mixed solution, mix gently, add in a hole of 6 orifice plates, hatch for 27 DEG C.
5) shift out transfection mixture, in Xiang Qikong, add the full Grace substratum 2ml containing foetal calf serum, amphotericin, penicillin and Streptomycin sulphate, after 27 DEG C of cultivation 84h, gather in the crops viral supernatants and cell respectively.
2. virus amplification
1.2 × 10 are inoculated in 125cm2 culturing bottle 7individual Sf9 cell, inside add Grace nutrient solution 30ml, cultivate 2 hours, make cell completely adherent for 27 DEG C, add provirus liquid (viral supernatants that previous step obtains) again in 1:10 ratio, cultivate 72h for 27 DEG C and gather in the crops supernatant, 4 DEG C of 5000g, centrifugal, remove cell precipitation, getting supernatant is virus liquid, and lucifuge 4 DEG C saves backup.
3. the mensuration of baculovirus titers:
Adopt the plaque analyses method that Invitrogen recommends.With 5 × 10 in 35mm disposable plastic culture dish 5cell/ml measures inoculation, after completely adherent, discard culture supernatant, cleans once with serum-free, antibiotic-free Grace substratum.By virus liquid serum-free, antibiotic-free TNM-FH from 10 -310 times are diluted to 10 successively -8, every hole 1m1 liquid, room temperature absorption 1h, inhales and abandons liquid.Heat fused is dropped to 2% low melting-point agarose (preparing with the ultrapure water) 1m1 of about 40 DEG C and equivalent 2 × maintenance medium (the Grace substratum containing serum) mixes, covers cell surface, note avoiding producing bubble.After agarose solidifies, put in incubator, be inverted for 27 DEG C and cultivate 4-10 days.Observe the generation of canescence plaque every day.Until plaque number no longer changes, look for hole (plate) counting having 3-20 plaque.
Calculate virus titer (pfu/ml), its calculation formula is: virus titer (pfu/ml)=1/ extension rate × every plaque number × 1/, hole (every hole inoculation ml number).MOI (multiplicity of infection) refers to infect the ratio between virus and infected cell, can calculate the viral liquid measure infected needed for a certain amount of cell accordingly with virus titer.Infect required viral liquid measure (ml)=[MOI value (the PFU/cell) × total cellular score of expectation]/virus liquid titre (PFU/ml).
Embodiment 4 protein expression and purifying
1. high MOI recombinate shape virus infection cell expressing albumen
By multiple 125cm2 culturing bottle mass propgation Sf9 cell, by each hypotype P4 for recombinant virus according to 1 ~ 10 MOI respectively infected insect cell.Collect the Sf9 cell infecting 72h, centrifugal 5 minutes of 5000g.
2. ultrasonic degradation insect cell
Under ice-water bath, by the cell of precipitation PBS buffer solution 2 times, by 1 × 10 7cell/ml adds PBS damping fluid again.Ultrasound intensity 40%, each 5s, interval 10s, altogether 90s.After ultrasonic in 4 ° of C with the centrifugal 40min of 10000g, collect supernatant and precipitation.
3. ultracentrifugation purifying HPV L1 albumen
In supernatant, with CsCl-PBS regulating density to 1.27g/ml, with SW40, with 31000rpm(Beckman, Optima L-100XP type ultracentrifuge) in 20 DEG C, centrifugal 24 hours.Liquid after 96 orifice plate Fractional Collectionses are centrifugal.Respectively get 1 μ L and be splined on 10%SDS-PAGE glue, after electrophoresis, carry out Coomassie brilliant blue dyeing and western blotting.Choose the collection liquid that density is 1.26-1.28g/ml, turn the sucrose density bed course of 45%, above it, carefully add the protein liquid after dialysis.4 DEG C, 35000rpm, 2.5 hours.Fractional Collections centrifugate, respectively gets 1 μ L and is splined on 10%SDS-PAGE glue, carries out Coomassie brilliant blue dyeing and western blotting (concrete grammar is with reference to " molecular cloning guide "), obtain 1-15 target protein sample after electrophoresis.
4. electron microscopic observation
By ultracentrifugation purifying HPV L1 albumen, through 2% phospho-wolframic acid negative staining, transmission electron microscope observing VLP form, diameter is about 55-60nm, and size is even, is rendered as hollow form.
Embodiment 5HPV L1 viruslike particle Immunity identification
1. the preparation of pseudovirus
By reporter plasmid (pcDNA3.1-Luc+) (purchased from Invitrogen) and structure gene plasmid (pShell6, pShell16, pShell18, pShell31, pShell45, pShell52 and pShell58) (Invitrogen) cotransfection 293FT cell (purchased from Invitrogen) respectively.With DMEM substratum (purchased from Invitrogen) harvested cell after 48 hours, draw a small amount of suspension simultaneously and carry out cell counting.Centrifugal 1500rpm, 5min; Abandon supernatant, add 3ml DPBS-Mg(Invitrogen) washed cell, centrifugal 1500rpm, 5min; Abandon supernatant, add 3mlDPBS-Mg(containing 9.5mmol/L Mgcl 2with the DPBS solution of 1 × fungal antibiotic) washed cell, draw 60 μ L cell suspensions and be stored in-80 DEG C and use in contrast.All the other samples are with the centrifugal 5min of 1500rpm, and abandon supernatant, cell precipitation is with 1 × 10 8the concentration of individual/ml is dissolved in cell pyrolysis liquid (the DPBS-Mg solution containing 0.5%Brij58,0.2%Benzonase, 0.2%Plasmid Safe), and generally the volume of cell precipitation can account for about 1/3 of final volume.Be placed in 37 DEG C of incubators at least 16 hours, make pseudovirus PsV change comparatively stable conformation into, by turning upside down, lysate being mixed every for some time, within particularly initial two hours, want frequent.Mixture is placed in 5 minutes on ice, adds the 5M NaCl of about 1/5 mixture cumulative volume, make NaCl ultimate density be 850mM.Mixture is placed in 10 ~ 20 minutes on ice.4 DEG C of centrifugal 1500rpm, 10min, stay supernatant ,-80 DEG C of preservations.
2. the ultracentrifugation purifying of pseudovirus
In super clean bench, add 40% sucrose solution 5ml in ultracentrifugation pipe.Slowly join gathering in the crops the pseudovirus DPBS-Mg polishing obtained in centrifuge tube to 5ml, be divided into two-layer up and down, upper strata is the DPBS-Mg solution containing pseudovirus, and lower floor is 40% sucrose solution.Centrifuge tube is placed in SW40 rotor, rotating speed 27500rpm, 3.5 hours time, temperature 4 degree.Abandon supernatant, carefully precipitation at the bottom of cylinder is hanged with DPBS-Mg, be stored in-80 DEG C.
3. pseudovirus virus titer measures
293FT cell being laid on 24(96 in 72 hours in advance) in the DMEM Tissue Culture Plate of hole, every porocyte number is 2 × 10 5individual/100 μ l, in 37 DEG C and 5%CO 2under cultivate in incubator.HPV16, HPV18, HPV52, HPV58, HPV6, HPV45, HPV31 pseudovirus is carried out 10 respectively 3, 10 4, 10 5with 10 6doubly dilution, each dilution dilution final volume of various pseudovirus is 200 μ l.In 24 porocyte culture plates, every hole adds 100 μ l pseudovirus diluents, and each extent of dilution of every type pseudovirus establishes 3 holes.After 72 hours, suck supernatant, every hole adds cell pyrolysis liquid 95 μ l, is blown afloat by every porocyte fast, is placed in EP pipe, 13200rpm, centrifugal 30s.Draw supernatant 80 μ l, add in 96 hole blanks (purchased from WHB company), add 20 μ l(10 μ l fast to each hole again) luciferase substrate, 96 hole blanks are placed in multi-functional microplate reader and detect.(experimental value-blank value)/(negative control value-blank value) >10 is judged to be that (blank value is that hollow plate records to the positive; Negative control only substratum records), calculate pseudovirus titre.
4. animal immune
6-8 week Ba1B/C female mice 8,18-20g, is divided into 2 groups at random: adopt abdomen injection, experimental group 5, often only injects 10gVLP, control group 3, every only injection PBS liquid 1ml, immunity amounts to 3 times, just exempts to carry out booster immunization the 2nd and 4 weeks with same dosage respectively afterwards, 14 days after final immunization, crane one and put to death mouse, heart extracting blood, after room temperature is solidified, collect VLP antiserum(antisera) ,-70 DEG C of preservations after packing.
5. serum antibody is to the experiment of the blocking effect of pseudovirus
Within 72 hours in advance, paving 293FT cell is in 24(96) in the DMEM Tissue Culture Plate of hole, every porocyte is 2 × 10 4individual/100 μ l, 37 DEG C, 5%CO 2cultivate in incubator.By HPV6, HPV11, HPV16, HPV18, HPV58 positive serum DMEM substratum dilutes 3200 times.。On 24 porocyte culture plates, pseudovirus pats Tissue Culture Plate surrounding after mixing in the ratio of 1:1 with immune serum, makes pseudovirus and serum mixing, two holes are established in often kind of combination, the control wells of simultaneously establishing negative serum to mix with pseudovirus and substratum control wells.Various mixture is placed in 1 hour on ice.Joined by mixture in orifice plate, after 72 hours, suck supernatant, every hole adds cell pyrolysis liquid 95 μ l, is blown afloat by every porocyte fast, is placed in EP pipe, 13200rpm, centrifugal 30 seconds.Draw supernatant 80 μ l, add in 96 hole blanks, add 20 μ l luciferase substrate again to each hole fast.96 hole blanks are placed in multi-functional microplate reader detect.
Infect blocking-up rate=(experimental value of pseudovirus experimental value ﹣ blocking aperture)/pseudovirus experimental value × 100%.
The definition of NAT: reach the maximum dilution multiple infecting blocking-up rate higher than 50%.More than 50% antibody infecting inhibiting rate can be reached and be regarded as that there is neutralising capacity after 50 times of dilutions.
Experimental result: table 3.
Table 3. NAT contrasts
Embodiment 6ELISA method measures protein expression
(1) each sample (1-15 protein sample) is got 1 μ l and is added PBS9 μ l and dilute, and therefrom respectively gets 1 μ l(PBS49 μ l and dilutes) add ELISA96 orifice plate respectively, hatch 16 hours for 4 DEG C.5% skim-milk-PBST(1L PBS+1mL Tween20) 50 μ l/ holes.Close 3 hours for 4 DEG C, abandon supernatant, PBS-T washes plate 3 times (every hole 350 μ l);
(2) primary antibodie: anti-16L1-MAb(camvir antibody; Purchased from American Neomarkers company) each 50 μ l/ holes of 5% skim-milk-PBST of 1:2000, room temperature effect 1h abandons supernatant, and PBST washes 9 times (every hole 350ul);
(3) two resist: sheep anti mouse-HRP(is purchased from the raw work in Shanghai) 1:50005% skim-milk-PBST50 μ l/ hole, room temperature 1h abandons supernatant, and PBS-T washes plate 3 times (every hole 350ul);
(4) develop the color: add 100 μ l/ hole OPD(purchased from the raw work in Shanghai)-0.03%H 2o 2substrate solution (purchased from the raw work in Shanghai), 37 DEG C hatch 30min;
(5) OD is surveyed: every hole adds 50 μ l stop buffers (purchased from the raw work in Shanghai), measures absorbance at 450nm wavelength place.
Experimental result: table 4.
Table 4. expression amount contrasts
Type 6L1-500 6L1-491 6L1-479
Expression amount 19mg/L 32mg/L 42mg/L
Type 11L1-501 11L1-492 11L1-480
Expression amount 27mg/L 45mg/L 38mg/L
Type 16L1-505 16L1-498 16L1-483
Expression amount 12mg/L 20mg/L 24mg/L
Type 18L1-507 18L1-498 18L1-474
Expression amount 17mg/L 25mg/L 31mg/L
Type 58L1-498 58L1-491 58L1-479
Expression amount 28mg/L 37mg/L 45mg/L

Claims (11)

1. part removes the human mammilla tumor virus L 1 albumen that C holds nuclear localization signal, and wherein said human papillomavirus is HPV6 type, HPV11 type, HPV16 type, HPV18 type or HPV58 type, and described L1 albumen is selected from:
C holds brachymemma 9,21 amino acid whose HPV6 type L1 albumen; Preferably this truncated protein has SEQ ID NO:1,2; Preferred SEQ ID NO:2;
C holds brachymemma 9,21 amino acid whose HPV11 type L1 albumen; Preferably this truncated protein has SEQ ID NO:3,4; Preferred SEQ ID NO:4;
C holds brachymemma 7,22 amino acid whose HPV16L1 albumen; Preferably this truncated protein has SEQ ID NO:5,6; Preferred SEQ ID NO:6;
C holds brachymemma 9,24 amino acid whose HPV18L1 albumen; Preferably this truncated protein has SEQ ID NO:7,8; Preferred SEQ ID NO:8;
C holds brachymemma 7,19 amino acid whose humanpapilloma virus 58 L1 proteins; Preferably this truncated protein has SEQ ID NO:9,10; Preferred SEQ ID NO:10.
2. a polynucleotide sequence for coding HPV L1 albumen according to claim 1, preferably, described polynucleotide sequence is through codon optimized design; More preferably, described polynucleotide sequence is the codon optimized design used according to insect cell; Preferably, wherein:
The sequence of described HPV L1 gene is SEQ ID NO:11 or 12; More preferably,
The sequence of described HPV L1 gene is SEQ ID NO:12;
The sequence of described HPV L1 gene is SEQ ID NO:13 or 14; More preferably,
The sequence of described HPV L1 gene is SEQ ID NO:14;
The sequence of described HPV L1 gene is SEQ ID NO:15 or 16; More preferably,
The sequence of described HPV L1 gene is SEQ ID NO:16;
The sequence of described HPV L1 gene is SEQ ID NO:17 or 18; More preferably,
The sequence of described HPV L1 gene is SEQ ID NO:18; Or
The sequence of described HPV L1 gene is SEQ ID NO:19 or 20; More preferably,
The sequence of described HPV L1 gene is SEQ ID NO:20.
3. one kind comprises the carrier of polynucleotide according to claim 2.
4. comprise a cell for carrier according to claim 3, preferably, described cell is insect cell; More preferably, described cell is selected from sf9, sf21, HighFive insect cell.
5. a composition, comprises L1 albumen according to claim 1; Or polynucleotide according to claim 2; Or carrier according to claim 3; Or cell according to claim 4.
6. a human papillomavirus viruslike particle, it comprises L1 albumen according to claim 1.
7. obtain a method for viruslike particle according to claim 6, be included in baculovirus-insect cells polynucleotide sequence according to claim 2, then the cracking supernatant of the L1 albumen containing described brachymemma carried out purification process.
8. a HPV vaccine, it comprises the HPV16 viruslike particle formed by HPV16L1 albumen of claim 6; More preferably, described vaccine also comprises and is selected from following at least one HPV viruslike particle: the HPV6 viruslike particle formed by HPV6L1 albumen of claim 6, the HPV11 viruslike particle formed by HPV11L1 albumen, the HPV18 viruslike particle formed by HPV18L1 albumen and the HPV58 viruslike particle formed by humanpapilloma virus 58 L1 protein.
9. a HPV vaccine, it comprises the HPV58 viruslike particle formed by humanpapilloma virus 58 L1 protein of claim 6; Preferably, described vaccine contains the HPV58 viruslike particle of the humanpapilloma virus 58 L1 protein with SEQ ID NO:10 protein sequence; Preferably, described vaccine also contains the HPV16 viruslike particle formed by HPV16L1 albumen of claim 6 and the HPV18 viruslike particle formed by HPV18L1 albumen; Preferably, HPV16 viruslike particle contains the HPV16L1 albumen with SEQ ID NO:6 protein sequence, and HPV18 viruslike particle contains the HPV18L1 albumen with SEQ ID NO:8 protein sequence.
10. a HPV vaccine, the HPV6 viruslike particle formed by HPV6L1 albumen that described vaccine contains claim 6 and the HPV11 viruslike particle formed by HPV11L1 albumen; Preferably, described vaccine contain the HPV6L1 albumen with SEQ ID NO:2 protein sequence HPV6 viruslike particle and containing the HPV11 viruslike particle of HPV11L1 albumen with SEQ ID NO:4 protein sequence; Preferably, described vaccine also contains the HPV16 viruslike particle formed by HPV16L1 albumen of claim 6 and the HPV18 viruslike particle formed by HPV18L1 albumen; Preferably, HPV16 viruslike particle contains the HPV16L1 albumen with SEQ ID NO:6 protein sequence, and HPV18 viruslike particle contains the HPV18L1 albumen with SEQ ID NO:8 protein sequence.
11. 1 kinds of HPV vaccines, the HPV58 viruslike particle that described vaccine contains the HPV6 viruslike particle formed by HPV6L1 albumen of claim 6, the HPV11 viruslike particle formed by HPV11L1 albumen and formed by humanpapilloma virus 58 L1 protein; Preferably, described vaccine contain the HPV6L1 albumen with SEQ ID NO:2 protein sequence HPV6 viruslike particle, containing have SEQ ID NO:4 protein sequence HPV11L1 albumen HPV11 viruslike particle and containing the HPV58 viruslike particle of humanpapilloma virus 58 L1 protein with SEQ ID NO:10 protein sequence; Preferably, described vaccine also contains the HPV16 viruslike particle formed by HPV16L1 albumen of claim 6 and the HPV18 viruslike particle formed by HPV18L1 albumen; Preferably, HPV16 viruslike particle contains the HPV16L1 albumen with SEQ ID NO:6 protein sequence, and HPV18 viruslike particle contains the HPV18L1 albumen with SEQ ID NO:8 protein sequence.
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