CN103694347A - Anti-human papilloma virus L1 protein antibody, and coding gene and application thereof - Google Patents
Anti-human papilloma virus L1 protein antibody, and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-human papilloma virus L1 protein antibody, and a coding gene and application thereof. The anti-human papilloma virus L1 protein antibody comprises a heavy chain variable region and a light chain variable region. The anti-human papilloma virus L1 protein antibody is characterized in that amino acid sequences of three hypervariable regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are respectively disclosed as SEQ ID NO.5-7; and amino acid sequences of three hypervariable regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are respectively disclosed as SEQ ID NO.8-10. The invention also discloses application of the anti-human papilloma virus L1 protein antibody in preparing reagents and medicines for detecting, preventing and treating human papilloma virus related diseases. The anti-human papilloma virus L1 protein antibody has high affinity with human papilloma virus L1 protein, and can be used for detecting and treating diseases of the human body.
Description
Technical field
The invention belongs to gene engineering technology field, relate in particular to a kind of anti-human papilloma virus (anti-HPV) L1 protein antibodies and encoding gene and application.
Background technology
Human papillomavirus (Human Papillomavirus, abbreviation HPV) be a kind of there is species specificity have a liking for epithelium virus, belong to the small DNA virus of double-stranded closed loop.HPV virus consists of this layer of coat of protein coat and the DNA core of the double-stranded circular being wrapped.Protein coat is comprised of main capsid protein (L1) and less important capsid protein (L2).1949, first Strauss observed HPV particle under Electronic Speculum, and it is spherical in shape, its 20 body symmetries, diameter 45-55nm.
The genome of HPV comprises approximately 8000 base pairs.HPV genome comprises 8 early stage open reading frames (Open Reading Frame is called for short ORF) (being E1--E8), 2 single open reading frames in late period (ORF) and 1 non-coding Chang Kong district.In open reading frame, E6 and E7 gene pairs Growth of Cells stimulate the most important in early days.E6, the E7 albumen of some HPV type E6, E7 coding of research discovery are combined with cancer suppressor gene p53 and Rb respectively, cause epithelial cell (as cervical epithelial cells) proliferation out of control.And late period reading frame L1 and L2 gene encode respectively main capsid protein and the less important capsid protein of HPV, be assembled into the capsid of HPV.
According to virus analysis, have been found that people's papillomatosis of more than 100 types.At present genome sequence definite HPV type approximately have more than 80 to plant, according to the position, epithelium place of its infection, be divided into skin-type HPV and reproductive tract epithelium HPV, about 35 kinds of types can infect female genital, approximately 20 kinds and Tumor-assaciated.According to different type HPV and tumorigenic dangerous height, be divided into low dangerous type and high-risk type HPV.Low dangerous type HPV comprises HPV6,11,42,43, the types such as 44, do not cause clinical symptom or cause tumour and the wart that the mankind are optimum, as be grown near reproductive organ mankind's verruca vulgaris, the pointed condyloma on skin and mucous membrane and be grown in papilloma on mucous membrane etc.; And high-risk type HPV comprises HPV16,18,31,33,35,39,45,51,52,56,58,59,66, the types such as 68, comprise the very high dependency that has, especially HPV16 and 18 types of tumour with infected epithelial cell height pathology.The human papillomavirus of these types is main relevant with the generation of reproductive tract malignant change, as cervical cancer, anus cancer, carcinoma of vagina and penile cancer.In addition, high-risk HPV is found relevant with some oral cavity and laryngocarcinoma.
It is a long-term process that HPV infects reproductive tract, can hide the several years in cell, once chance ripe (as host's immunity of organisms reduces), the virus of hiding can reactivate.HPV course of infection is divided into latent infection phase, subclinical infection phase, tumor stage that clinical stage is relevant with HPV conventionally.Cervical cancer also has a series of precursor lesion, it is epithelium of cervix uteri atypical hyperplasia, on pathology, claim cervical intraepithelial neoplasia to become (cervical intraepithelial neoplasia, abbreviation CIN), conventionally according to severity, be divided into three grades again: in epithelium of cervix uteri, slight knurl becomes (CIN I), the interior moderate knurl of epithelium of cervix uteri becomes (CIN II) and epithelium of cervix uteri inner height knurl becomes (CIN III), and these precancerous lesions all likely develop into invasive carcinoma of cervix.
The course of infection of HPV has its singularity.The breeding of HPV virus often only limits in the squamous cell in end differentiation, in addition, HPV virus can postpone human body it is produced to immune response [Schill JT within the long duration, Day PM, Kines R (2010) Current understanding of the mechanism of HPV infection.Gynecol.Oncol.118:S12-S17.].The tremendous development of HPV course of infection research has benefited from the progress of modern molecular biology in recent years.Kirnbauer etc. prove [Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT. (1992) Papillomavirus L1major capsid protein self-assembles into virus-like particles that are highly immunogenic.Proc Natl Acad Sci USA.89:12180 – 12184.] main capsid protein (L1 albumen) single expression or all can be self-assembled into without infective empty capsid sample particle (virus-like-particle with less important capsid protein (L2 albumen) coexpression, be called for short VLP), and there is very high immunogenicity.VLP can be is effectively surperficial acceptor [the Roden RB that combines with cell by multiple epithelial cell and cultured cells, Kirnbauer R, Jenson AB, Lowy DR, Schiller JT. (1994) Interaction of papillomaviruses with the cell surface.J Virol.68:7260 – 7266.].This VLP structure is similar to natural virion, has the antigen predicting space epitope identical with intact virus, can infection induced animal model or human body produce the neutralizing antibody of high titre, to protect body to avoid infecting.
This characteristic of L1 capsid protein provides good means [Suzich JA for developing preventative HPV vaccine; Ghim SJ; Palmer-Hill FJ; White WI; Tamura JK; Bell JA, et al. (1995) Systemic immunization with papillomavirus L1protein completely prevents the development of viral mucosal papillomas.Proc Natl Acad Sci USA.92:11553 – 11557; Kirnbauer; R; Chandrachud LM, O ' Neil BW, Wagner ER; Grindlay GJ; Armstrong A, McGarvie GM, Schiller JT; D.R.Lowy, Campo MS. (1996) Virus-like particles of bovine papillomavirus type4in prophylactic and therapeutic immunization.Virology219:37 – 44; With Schiller JT, Lowy DL. (2006) Prospects for cervical cancer prevention by human papillomavirus vaccination.Cancer Res.66:10229 – 10232].
The antibody that VLP induction produces can prevent infection [the Breitburd FKR of papilloma virus effectively; Hubbert NL; Nonnenmacher B; Trin-Dinh-Desmarquet C; Orth G; Schiller JT, et al. (1995) Immunization with virus-like particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection.J Virol.69:3959 – 3963; With Suzich JA, Ghim SJ, Palmer-Hill FJ, White WI, Tamura JK, Bell JA, et al. (1995) Systemic immunization with papillomavirus L1protein completely prevents the development of viral mucosal papillomas.Proc Natl Acad Sci USA.92:11553 – 11557].Research shows, the monoclonal antibody of anti-VLP also can be effectively in and HPV virus to the infectivity of people's cell [Day PM, Thompson CD, Buck CB, Pang YY, Lowy DR, Schiller JT. (2007) Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition.J Virol.81:8784 – 8792.]
In addition, there are some researches show that detect HPV L1 albumen also has an effect [Jia Yongcun in the diagnosis and prognosis of uterine neck human papillomaviral infection is judged, Fan Yang, Na Wenxia (2010) HPV L1 detects the effect in the prognosis of uterine neck human papillomaviral infection is judged.< < Ningxia medical journal > > 8:688-690]; [Qi Ruiling, Jia Xiaoyun, Tang Siyuan (2012) human papillomavirus L1 glutelin detects cervical disease diagnostic value. < < China practical diagnosis and treatment magazine > > 26 (8): 785-786]; [history is strong, Deng Kuanguo, Liu Yuxia, Yin Shihua (2010) human papillomavirus and the application of L1 Protein Detection in diagnosis of cervical lesion thereof.Medical test and clinical .21 (6): 70-76].
Therefore, obtain detect and in the disease that causes for test-and-treat HPV with the total man source anti-L1 capsid protein antibody of human papillomavirus seem very meaningful.
Summary of the invention
The invention provides a kind of anti-human papilloma virus (anti-HPV) L1 protein antibodies, this antibody is total man source anti-human papilloma virus (anti-HPV) L1 protein antibodies, has high-affinity with human mammilla tumor virus L 1 albumen (main capsid protein), and specificity is good.
An anti-human papilloma virus (anti-HPV) L1 protein antibodies, comprises variable region of heavy chain and variable region of light chain, and three hypervariable region CDRH1, CDRH2 of described variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively:
gDSVSSNSNTA,
yYRSKWY,
iIAPDAFDI;three hypervariable region CDRL1, CDRL2 of described variable region of light chain, the aminoacid sequence of CDRL3 are respectively
rASQSISGWLA,
gASTLES,
qQYNNYPWT.
CDRH1 is positioned at 26th～36 of variable region of heavy chain, and aminoacid sequence is as shown in SEQ ID NO.5; CDRH2 is positioned at 56th～62 of variable region of heavy chain, and aminoacid sequence is as shown in SEQ ID NO.6, and CDRH3 is positioned at 104th～112 of variable region of heavy chain; Aminoacid sequence is as shown in SEQ ID NO.7.
CDRL1 is positioned at 24th～34 of variable region of light chain, and aminoacid sequence is as shown in SEQ ID NO.8; CDRL2 is positioned at 50th～56 of variable region of light chain, and aminoacid sequence is as shown in SEQ ID NO.9; CDRL3 is positioned at 89th～97 of variable region of light chain, and aminoacid sequence is as shown in SEQ ID NO.10.
On antibody molecule variable region, there is very large variability in the aminoacid sequence of three hypervariable regions, and the region aminoacid sequence between hypervariable region changes less.These three hypervariable regions can form accurate complementation with antigenic determinant on space structure, the complementary determining region (CDR) that is therefore otherwise known as, and the CDR of different heavy chains, light chain has determined the specificity of antibody to antigen.
Preferably, the aminoacid sequence of described variable region of heavy chain is as shown in SEQ ID No.1, and the aminoacid sequence of described variable region of light chain is as shown in SEQ ID No.2.
Described anti-human papilloma virus (anti-HPV) L1 protein antibodies can be the antigen-binding portion thereof of whole antibody or whole antibody.Described whole antibody is preferably IgG1 type; Described antigen-binding portion thereof is preferably Fab fragment, Fab ' fragment, F (ab ')
2fragment or single-chain antibody, more preferably single-chain antibody.
Antigen-binding portion thereof had both retained the region that can be combined with antigen-specific, had avoided again the antigenicity of Fc fragment and the side effect that causes.Wherein, single-chain antibody has in easy infiltration tumor tissues increases drug level, and immunogenicity is little, in vivo the transformation period of circulation short, be easy to remove, thereby be easy to be connected with toxin or enzyme gene the advantages such as direct adaptive immune toxin or enzyme labelled antibody.
Antigen-binding portion thereof can be prepared by DNA recombinant technology or by enzymatic/chemical cracking whole antibody.The preparation method of anti-human papilloma virus (anti-HPV) L1 albumen single-chain antibody of the present invention is: adopt the disclosed method of Chinese patent literature that publication number is CN1444651A to build people's single-chain antibody library, and screen anti-human papilloma virus (anti-HPV) L1 protein antibodies in this people's single-chain antibody library.
The present invention also provides the gene of the described anti-human papilloma virus (anti-HPV) L1 protein antibodies of coding, and wherein, the nucleotide sequence of encoding heavy chain variable region gene is as shown in SEQ ID No.3, and the nucleotide sequence of encoded light chain variable region gene is as shown in SEQ ID No.4.
The present invention also provides recombinant vectors or the expression system that contains described encoding gene.The initial carrier of described recombinant vectors is pACT2 or pET27b.
The application of anti-human papilloma virus (anti-HPV) L1 protein antibodies described in the present invention also provides in the medicine of preparation prevention, treatment human papillomavirus relative disease.
The application of anti-human papilloma virus (anti-HPV) L1 protein antibodies described in the present invention also provides in the reagent of preparation detection human papillomavirus relative disease.
Anti-human papilloma virus (anti-HPV) L1 protein antibodies of the present invention can, specifically in conjunction with human mammilla tumor virus L 1 albumen, detect the cell that HPV infects.Wherein, utilize the human-derived anti-human papilloma virus L1 protein antibodies of total length restructuring can detect the cell that HPV infects, therefore, can utilize anti-human papilloma virus (anti-HPV) L1 protein antibodies of the present invention to prepare the reagent that detects human papillomavirus relative disease.
In addition, research finds, HPV antibody can be effectively in and the infectivity of HPV virus to people's cell, therefore, can utilize the anti-human papilloma virus (anti-HPV) L1 protein antibodies of high-affinity of the present invention to prepare medicine, to prevent, to treat human papillomavirus relative disease.
Wherein, in above-mentioned two kinds of application, described human papillomavirus relative disease can be for any disease that infects relevant (directly or indirectly causing) with human papillomavirus (HPV), as cervical cancer, anus cancer, carcinoma of vagina, penile cancer, oral carcinoma, laryngocarcinoma etc.Further, described human papillomavirus relative disease is specially cervical intraepithelial neoplasia change or the cervical cancer that human papillomavirus causes, further, described human papillomavirus relative disease can be the disease relevant to 18 type human papilloma virus infections.
Compared with prior art, beneficial effect of the present invention is:
Anti-human papilloma virus (anti-HPV) L1 protein antibodies of the present invention is to infect for detection of HPV, also can be for the total man source anti-human papilloma virus (anti-HPV) L1 protein antibodies of prevention, treatment human body diseases, avidity is high, energy specific combination human mammilla tumor virus L 1 albumen, therefore, can be made into medicine or reagent, effectively detect, prevent and treat HPV and infect the disease causing.
Accompanying drawing explanation
Fig. 1 is the structural representation of single-chain antibody of the present invention.
Fig. 2 is that the present invention is at the expressed blue coloration result of HPV 18 L1 albumen Coomassie after SDS-PAGE of insect sf9 cell; Wherein, the 1st road: sf9 total protein of cell after empty carrier infects; The 2nd road: the carrier containing HPV18-L1 infects rear sf9 total protein of cell; The 3rd road: the HPV18 type L1 albumen after purifying.
Fig. 3 is the ELISA detected result of anti-human papilloma virus (anti-HPV) L1 albumen single-chain antibody #H18L1-A of the present invention.
Fig. 4 is the Western Blot result of anti-human papilloma virus (anti-HPV) L1 protein antibodies YFH18L1-A of the present invention; Wherein, the 1st road: normal cervix cell sample; The 2nd road: CIN2 phase patient's cervical exfoliated cell; The 3rd road: the cervical exfoliated cell of cervical cancer patient.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
The screening of embodiment 1 anti-HPV 18 (HPV18) L1 albumen single-chain antibody
Adopt the disclosed method of Chinese patent literature that publication number is CN1444651A to build people's single-chain antibody library, and in this people's single-chain antibody library, screen anti-HPV 18 (HPV18) L1 albumen single-chain antibody, specific implementation process is as follows:
1 amplification obtains human antibody heavy chain and variable region of light chain DNA
The poly A+RNA(of take from people's marrow, people's tire liver, people's spleen and human peripheral leucocytes is purchased from Clontech) be template, utilize oligo(dT) and random primer (random primers), use reverse transcriptase test kit (purchased from Clontech), the Methods Instruction providing according to Clontech test kit, by the reverse cDNA that is transcribed into of poly A+RNA.
Take above-mentioned cDNA as template, utilize the primer of a series of identification human antibody heavy chain variable regions (VH) and variable region of light chain (VL) gene, carry out the DNA sequence dna that pcr amplification obtains variable region of heavy chain and variable region of light chain all in people's antibody.The primer sequence of a series of identification human antibody heavy chains and chain variable region gene is as follows:
First group of 5 '-end primer (SEQ ID No.11～17) for amplification human antibody heavy chain variable region (VH) gene, comprising:
VH1b：5’-
CCATACGATGTTCCAGATTACCAGGTGCAGCTGCAGGAGTC(C/G)G-3’；
VH2b：5’-
CCATACGATGTTCCAGATTACCAGGTACAGCTGCAGCAGTCA-3’；
VH3b：5’-
CCATACGATGTTCCAGATTACCAGGTGCAGCTACAGCAGTGG?G-3’；
VH4b：5’-
CCATACGATGTTCCAGATTACGAGGTGCAGCTG(G/T)TGGAG(A/T)C(C/T)-3’；
VH5b：5’-
CCATACGATGTTCCAGATTACCAGGTCCAGCT(G/T)GT(A/G)CAGTCTGG-3’；
VH6b：5’-
CCATACGATGTTCCAGATTACCAG(A/G)TCACCTTGAAGGAGTCTG-3’；
VH7b：5’-
CCATACGATGTTCCAGATTACCAGGTGCAGCTGGTG(C/G)A(A/G)TCTGG-3’；
Second group of 3 '-end primer (SEQ ID No.18～23) for amplification human antibody heavy chain variable region (VH) gene, comprising:
VH1f：5’-
GCCGCCTGATCCACCACCGCCTGAGGAGAC(A/G)GTGACCAGGGTG-3’；
VH2f：5’-
GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCAGGGTT-3’；
VH3f：5’-
GCCGCCTGATCCACCACCGCCTGAAGAGACGGTGACCATTGT-3’；
VH4f：5’-
GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCGTGGTCC-3’；
VH5f：5’-
GCCGCCTGATCCACCACCGCCGGTTGGGGCGGATGCACTCC-3’；
VH6f：5’-
GCCGCCTGATCCACCACCGCC(C/G)GATGGGCCCTTGGTGGA(A/G)GC-3’；
The 3rd group of 5 '-end primer (SEQ ID No.24～32) for amplification people's antibody λ-variable region of light chain (V λ) gene, comprising:
VL1b：5’-
GGCAGCGGTGGTGGAGGCAGTCAGTCTGT(C/G)(C/G/T)TGACGCAGCCGCC-3’；
VL2b：5’-
GGCAGCGGTGGTGGAGGCAGTTCCTATG(A/T)GCTGAC(A/T)CAGCCAC-3’；
VL3b：5’-
GGCAGCGGTGGTGGAGGCAGTTCCTATGAGCTGA(C/T)(A/G)CAGC(C/T)ACC-3’；
VL4b：5’-
GGCAGCGGTGGTGGAGGCAGTCAGCCTGTGCTGACTCA(A/G)(C/T)C-3’；
VL5b：5’-
GGCAGCGGTGGTGGAGGCAGTCAG(A/G/T)CTGTGGTGAC(C/T)CAGGAGCC-3’；
VL6b：5’-
GGCAGCGGTGGTGGAGGCAGTCAGCC(A/T)G(G/T)GCTGACTCAGCC(A/C)CC-3’；
VL7b：5’-
GGCAGCGGTGGTGGAGGCAGTTCCTCTGAGCTGA(C/G)TCAGGA(C/G)CC-3’；
VL8b：5’-
GGCAGCGGTGGTGGAGGCAGTCAGTCTG(C/T)(C/T)CTGA(C/T)TCAGCCT-3’；
VL9b：5’-
GGCAGCGGTGGTGGAGGCAGTAATTTTATGCTGACTCAGCCCC-3’；
The 4th group of 3 '-end primer (SEQ ID No.33～34) for amplification people's antibody λ-variable region of light chain (V λ) gene, comprising:
VL1f：5’-
GGGGTTTTTCAGTATCTACGATAGGACGGT(C/G)A(C/G)CTTGGTCC-3’；
VL2f：5’-
GGGGTTTTTCAGTATCTACGAGAGGACGGTCAGCTGGGTGC-3’；
The 5th group of 5 '-end primer (SEQ ID No.35～38) for amplification people antibody k-variable region of light chain (Vk) gene, comprising:
VK1b：5’-
GGCAGCGGTGGTGGAGGCAGTGACATCC(A/G)G(A/G/T)TGACCCAGTCTCC-3’；
VK2b：5’-
GGCAGCGGTGGTGGAGGCAGTGAAATTGT(A/G)(A/T)TGAC(A/G)CAGTCTCC-3’；
VK3b：5’-
GGCAGCGGTGGTGGAGGCAGTGATATTGTG(A/C)TGAC(C/G/T)CAG(A/T)CTCC-3’；
VK4b：5’-
GGCAGCGGTGGTGGAGGCAGTGAAACGACACTCACGCAGTCTC-3’；
The 6th group of 3 '-end primer (SEQ ID No.39～42) for amplification people antibody k-variable region of light chain (Vk) gene, comprising:
VK1f：5’-
GGGGTTTTTCAGTATCTACGATTTGATTTCCACCTTGGTCC-3’；
VK2f：5’-
GGGGTTTTTCAGTATCTACGATTTGATCTCCA(C/G)CTTGGTCC-3’；
VK3f：5’-
GGGGTTTTTCAGTATCTACGATTTGATATCCACTTTGGTCC-3’；
VK4f：5’-
GGGGTTTTTCAGTATCTACGATTTAATCTCCAGTCGTGTCC-3’。
During variable region of heavy chain (VH) in amplification people antibody, utilize the combination of first group of primer and second group of primer, have 42 PCR to react; During λ-variable region of light chain (V λ) in amplification people antibody, utilize the combination of the 3rd group of primer and the 4th group of primer, have 18 PCR to react; During k variable region of light chain (Vk) in amplification people antibody, utilize the combination of the 5th group of primer and the 6th group of primer, have 16 PCR to react.
In first group of primer, include yeast two-hybrid carrier pACT2(Hua SB, Luo Y, Qiu M, Chan E, Zhou H, Zhu L. (1998) Gene.215:143-152.) (Hua SB, Qiu M, Chan E, Zhu L, Luo Y. (1997) Plasmid.38:91-96.) the upstream homologous sequence (underscore part) of multiple clone site; The downstream homologous sequence (underscore part) that includes yeast two-hybrid carrier pACT2 multiple clone site in the 4th group and the 6th group of primer; And including connection peptides sequence (underscore part) in second group, the 3rd group and the 5th group of primer, connection peptides is for connecting variable region of heavy chain and the variable region of light chain of antibody.
During amplification, PCR reaction system is all identical with reaction conditions, and PCR reaction system is:
Above-mentioned each component is mixed to be placed in PCR instrument and react.Reaction conditions is: 94 ℃ are unwind 1 minute, anneals 1 minute for 50 ℃, and 72 ℃ are extended 2.5 minutes, circulate 30 times.
The homologous sequence of 2 connection carrier multiple clone site and connection peptides sequence
(1) take the human antibody heavy chain variable region DNA of PCR gained in step 1 is template, take respectively primer 7 and primer 8 as upstream primer and downstream primer, and sequence is:
Upstream primer (underscore is partly the upstream homologous sequence of carrier pACT2 multiple clone site for primer 7, SEQ ID No.43):
5’-ACCCCACCAAACCCAAAAAAAGAGATCTGTATGGCT
TACCC ATACGATGTTCCAGATTAC-3’；
Downstream primer (underscore is partly connection peptides anti-chain sequence for primer 8, SEQ ID No.44):
5’-ACTGCCTCCACCACCGCTGCCACCTCCGCCAGATCCTCC
GCC GCCTGATCCACCACCGCC-3’；
Carry out pcr amplification, reaction conditions and system are with step 1.Reacted rear acquisition containing the upstream homologous sequence of 5 '-carrier pACT2 multiple clone site and the human antibody heavy chain variable region DNA sequence dna of 3 '-connection peptides anti-chain sequence.
(2) take the human antibody light chain variable region DNA of PCR gained in step 1 is template, with primer 9 and primer 10, is respectively downstream primer and upstream primer, and sequence is as follows:
Upstream primer (underscore is partly that connection peptides is along chain-ordering for primer 10, SEQ ID No.45):
5’-GGCGGTGGTGGATCAGGCGGCGGAGGATCTGGCGGAGGT
GG CAGCGGTGGTGGAGGCAGT-3’；
Downstream primer (underscore is partly the downstream homologous sequence of carrier pACT2 multiple clone site for primer 9, SEQ ID No.46):
5’-GAGATGGTGCACGATGCACAGTTGAAGTGAACTTGC
GGGGT TTTTCAGTATCTACGA-3’；
Carry out pcr amplification, reaction conditions and system are with step 1.Reacted rear acquisition containing the downstream homologous sequence of 3 '-carrier pACT2 multiple clone site and 5 '-connection peptides the human antibody light chain variable region DNA sequence dna along chain-ordering.
3 connect single-chain antibody
What pcr amplification in step 2 was obtained mixes containing the homologous sequence of carrier pACT2 multiple clone site and DNAHe variable region of light chain, the human antibody heavy chain variable region DNA of connection peptides sequence, take this hybrid dna as template, take primer 7 and primer 9 respectively as upstream primer and downstream primer, carry out pcr amplification, reaction conditions and system are with step 1.
As shown in Figure 1, obtain comprising single-chain antibody (scFv) DNA of human antibody heavy chain variable region DNA sequence dna, variable region of light chain DNA sequence dna, carrier pACT2 multiple clone site homologous sequence and connection peptides DNA sequence dna after having reacted.
4 build people's single-chain antibody gene library
The method (Yeast Protocol Handbook, PT3024-1) that the single-chain antibody that step 3 is obtained (scFv) DNA and yeast two-hybrid carrier pACT2 after restriction enzyme (Bam HI and Eco RI) is processed provide according to former Clontech company proceeds to yeast strain Y187(MAT α, ura3-52 jointly, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,112, gal4 Δ, gal80 Δ, met-, URA3::GAL1
uAS-GAL1
tATA-lac Z, MEL1) in, after homologous recombination in cell, single-chain antibody DNA is incorporated on pACT2 carrier, thereby obtains yeast two-hybrid single-chain antibody library, Gal4 active region on single-chain antibody DNA fragmentation and pACT2 carrier (Activation Domain, AD) merges.
For checking the quality in this single-chain antibody gene library, therefrom chosen at random 21 clones, Insert Fragment is carried out to sequencing analysis.Analytical results shows, all clones comprise the single-chain antibody DNA fragmentation merging with Gal4, and all single-chain antibody DNA sequence dnas are all unique.
Antibody dna copy number nearly 1 * 10 in the yeast two-hybrid single-chain antibody gene library obtaining through homologous recombination
8individual, can be applicable to yeast-two hybrid technique and screen specific antibody.
5 screening antibodies
(1) antibody screening
Use HPV 18 (HPV18) L1 albumen (capsid major protein) as antigen, antibody screening is carried out in yeast two-hybrid single-chain antibody gene library.
The DNA(sequence of encoding human papilloma virus HPV18 type L1 albumen is shown in to the ID X05015 of GenBank) recombinate in carrier pGBKT7, build pGBK-H18L1.PGBK-H18L1 coding Gal4DNA calmodulin binding domain CaM (Binding Domain is called for short BD), and merged human papillomavirus HPV18 type L1 albumen at its C end.
After the DNA sequence dna of encoding human papilloma virus HPV18 type L1 albumen is verified, pGBK-H18L1 plasmid DNA is transformed into yeast strain AH109(MATa, trp1-901, leu2-3,112, ura3-52, his3-200, gal4 Δ, gal80 Δ, LYS2::GAL1
uAS-GAL1
tATA-HIS3, GAL2
uAS-GAL2
tATA-ADE2, URA3::MEL1
uAS-MEL1
tATA-lac Z); AH109 yeast with pGBK-H18L1 plasmid can be in the upper growth of the synthetic medium (SD/-W) that does not contain tryptophane.
The MATa type yeast cell (AH109 bacterial strain) containing pGBK-H18L1 of equivalent and the MAT α type yeast cell (Y187 bacterial strain) containing single-chain antibody gene library are carried out to co-cultivation, make this two types cell mating combination.Owing to being loaded with the pACT2 carrier in single-chain antibody gene library, contain Leu2 gene, and pGBK-H18L1 comprises Trp1 gene, therefore, the yeast cell that comprises two kinds of plasmids can be grown in not the yeast synthetic medium (SD/-LW) containing leucine and Serine.
Exist the yeast cell of single-chain antibody scFv and L1 protein-interacting can activate reporter gene ADE2 and the HIS3 being incorporated in strain gene group, thereby make yeast cell can be grown in shortage VITAMIN B4, Histidine, the substratum of leucine and tryptophane (SD/-AHLW) is upper, and forms bacterium colony on this plate culture medium.
(2) specificity antibody screening
1) beta galactosidase enzyme analysis
Due to another reporter gene lac Z that exists the cell of scFv and L1 protein-interacting also can activate to be incorporated in strain gene group, therefore detect beta galactosidase enzyme in yeast cell and whether express the existence that can judge scFv/L1 albumen in yeast cell.
In screening culture medium (SD/-AHLW), altogether pick out 52 bacterium colonies, use beta galactosidase enzyme detection method to detect lac Z and express.Detection method is as follows:
1. will exist the yeast-inoculated of scFv/L1 protein-interacting to grow to screening culture medium (SD/-AHLW) flat board;
2. yeast colony is transferred on No. five filter paper of Whatman;
3. the filter paper with yeast cell bacterium colony is immersed among liquid nitrogen, make cell rupture;
4. filter paper is taken out from liquid nitrogen, and be placed in the baking oven of 30 ℃; Repeating step (3) and (4) twice;
5. filter paper is laid in appropriate X-gal solution, and is placed in the incubator of 37 ℃ approximately 15 minutes; If bacterium colony place shows blue, be indicated as the beta galactosidase enzyme positive, i.e. the lac Z gene expression that is activated.
Wherein, X-gal solution formula is as follows:
16.1g/L?Na
2HPO
4·7H
2O；
5.50g/L?NaH
2PO
4·H
2O；
0.75g/L?KCl；
0.246g/L?MgSO
4·7H
2O；
35mg/L?X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)；
5mM beta-mercaptoethanol; PH7.0.
Detected result: in above-mentioned 52 bacterium colonies picking out, have 23 to be that beta galactosidase enzyme is positive, show that reporter gene lacZ has been activated in these bacterium colonies.
2) specific combination analysis
ScFv to the positive bacterium colony of above-mentioned 23 beta galactosidase enzymes carries out specificity analyses, with verify single-chain antibody scFv whether specifically with the L1 protein binding of human papillomavirus HPV18 type.
From the yeast of above-mentioned 23 beta galactosidase enzyme positives, extract the pACT2 plasmid DNA that contains scFv, be more jointly transformed in AH109 yeast cell with pGBKT7 empty carrier DNA, pGBKT-IR α plasmid DNA, pGBKT-Lam plasmid DNA (coding Gal4DNA calmodulin binding domain CaM also merges and has human nuclear fabric layer albumen C at its C end) respectively; Yeast cell after conversion is first laid on SD/-LW plate culture medium grows, and then transfers on SD/-AHLW plate culture medium; The bacterium colony growing is done to beta galactosidase enzyme analysis, be verified as positive bacterium colony and be considered as containing nonspecific scFv, by its rejecting.
After above-mentioned specificity analyses, obtain one human mammilla tumor virus L 1 albumen is had to specific single-chain antibody, be numbered #H18L1-A, use ABI automatic sequencer to carry out sequencing analysis to this single-chain antibody, result shows:
The DNA sequence dna of the variable region of heavy chain of #H18L1-A is as shown in SEQ ID No.3.
The aminoacid sequence of the variable region of heavy chain of #H18L1-A (SEQ ID No.1) is:
QVQLQQSGPGLVKPSQTLSLTCVIS
GDSVSSNSNTAVWNWIRQSPSRGLEWLGRT
YYRSKWYQDYAVSVRSRISINPDTSKNQVSLQLTSVTPEDTAVYYCTK
IIAPDAFDIWGQGTMVTVSS；
Underscore is partly followed successively by three hypervariable region CDRH1, CDRH2, CDRH3(SEQ ID No.5～7);
The DNA sequence dna of the variable region of light chain of #H18L1-A as shown in SEQ ID No.4,
The aminoacid sequence of the variable region of light chain of #H18L1-A (SEQ ID No.2) is:
DIRLTQSPSTLSASVGDRVTITC
RASQSISGWLAWYQQRPGRAPKLLIY
GASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATFYC
QQYNNYPWTFGPGTKVEIK；
Underscore is partly followed successively by three hypervariable region CDRL1, CDRL2, CDRL3(SEQ ID No.8～10).
The preparation of recombinant human papilloma virus HPV18 type L1 albumen is with reference to [Peng Qinglin, Zhang Ting, Fan Dongsheng, Zheng Wei, Xie Xixiu, Xu Yufei, Xu Xuemei (2009) " expression and purification of HPV18L1 virus-like particle and the preparations of guinea pig antiserum thereof such as Peng Qinglin." preclinical medicine and clinical, 29:1039-1043].Briefly: the codon that HPV18 type L1 coding DNA fragment is adjusted to HPV18 type L1 coding DNA according to the codon frequency of sf9 insect cell is optimized, and the coding DNA after optimization inserts in pFastBac Dual carrier Xba I site and obtains recombinant vectors pFastBac-HPV18L1.By transforming intestinal bacteria DH10Bac competent cell, obtain restructuring Bacmid.With restructuring Bacmid transfection insect sf9 cell, in 27 ℃ of constant incubators, cultivate collecting cell culture supernatant after 72 hours, obtain the recombinant baculovirus of expressing HPV18 type L1 albumen.Utilize Plaque Technique Detected to measure after virus titer, with MOI=0.1 cells infected, carry out viral enrichment.Subsequently, with the virus liquid of high titre, infect sf9 insect cell, SDS-PAGE analyzes (see figure 2).
Recombinant virus infects sf9 cell in a large number, after 72h, collecting infecting cell, ultrasonication, 4 ℃, centrifugal 10 minutes of 12000rpm, get supernatant liquor, through CsCl Density ultracentrifugation purifying VLP[Shi W, Liu J, (2001) Papillomavirus pseudovirus:a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses.J Virol75:10139-10148. such as Huang Y].Finally use dialyzate (10mM HEPES, 150mM NaCl) dialysis within 3 hours, to obtain the HPV18 type L1 albumen (see figure 2) of purifying.
The preparation of the anti-HPV18 type of rabbit L1 serum is according to traditional method [the Harlow E of Harlow etc., with Lane D (1988) Antibodies:A Laboratory Manual.Cold Spring Harbor Laboratory press, the 53rd page to the 138th page].Get after HPV18L1 albumen after purifying is diluted to 400 microlitres and fully mix emulsification with equal-volume freund's adjuvant, respectively at 0,4,8 and 12 week subcutaneous multi-point injection immune rabbit.Within 1 week after last immunity, carry out venous blood sampling, ELISA detects serum antibody titer, and titre is got blood after reaching expection, collects serum.
1 single-chain antibody expression and purification
The encoding gene of single-chain antibody #H18L1-A is cloned in expression vector pET27b (+), builds and obtain pET27b-H18L1a;
PET27b-H18L1a is transformed into and expresses bacterium E.coli BL21 (DE3), and the method IPTG(0.5mM providing according to Novagen company) abduction delivering; In the target protein giving expression to, the N of scFv end is pelB sequence, and pelB sequence can be secreted into the scFv after expressing in the pericentral siphon chamber (periplasmic space) of BL21 (DE3); The C end of scFv contains a HSV marker and 6 * His marker, facilitates the purifying of target protein.
The method that application Qiagen company provides obtains the single-chain antibody of anti-human papilloma virus (anti-HPV) HPV18 type L1 albumen easily with Ni-NTA column separating purification.
The specificity of 2ELISA test single-chain antibody to HPV18 type L1 albumen
HPV18 type L1 albumen after sf9 insect cell expression purifying is shown in above-described embodiment 2.
ELISA testing method is as follows:
(1) with the coated 96-orifice plate of HPV18 type L1 albumen, at 2-8 ℃, spend the night;
(2) the 96-orifice plate after coated is made sealing treatment with SuperBlock again;
(3) single-chain antibody #H18L1-A is done in 0.02%BSA after serial dilution, add in the 96-hole that is coated with HPV18L1 albumen, with HPV18L1 protein binding;
(4) after 96-orifice plate is cleaned, add the antibody of the mouse-anti HSV marker of 5000 times of dilutions, be used for detecting the single-chain antibody combining;
(5), after 96-orifice plate is cleaned, add goat dynamics-horseradish peroxidase thing of 10000 times of dilutions;
(6), by after the final cleaning of 96-orifice plate, application horseradish peroxidase substrate TMB reagent is made color development treatment;
(7) use the sulfuric acid termination reaction of 0.5M, and detect the absorption spectrum of 450nm.
Detected result as shown in Figure 3, shows that single-chain antibody #H18L1-A can be effectively in conjunction with HPV18 type L1 albumen.
Embodiment 4 anti-human papilloma virus (anti-HPV) L1 protein antibodies detect HPV and infect cervical exfoliated cell
Entrust Yi Qiao Divine Land, Beijing Bioisystech Co., Ltd to prepare total length recombination human source anti-human papilloma virus (anti-HPV) L1 protein antibodies YFH18L1-A.YFH18L1-A is IgG1 type, and its variable region sequences is identical with the variable region sequences of single-chain antibody #H18L1-A.
The cervical exfoliated cell of normal cervix cell sample, cervical intraepithelial neoplasia (CIN) II phase (being the CIN2 phase) and cervical cancer patient is all from Hospital for Gynaecology and Obstetrics Attached to Medical Hospital of Zhejiang, and obtains patient's informed consent.
Western Blot analyzes total length human-derived anti-human papilloma virus HPV18 type L1 protein antibodies.Approximately 10
4cervical exfoliated cell is loaded onto 10% SDS-PAGE gel electrophoresis system after SDS-PAGE sample liquid is processed.After SDS-PAGE electrophoresis, protein delivery to nitrocellulose filter.Then, this for film 3% skim-milk-phosphoric acid buffer (pH7.4) sealing after 30 minutes, add the total length recombination human source anti-human papilloma virus (anti-HPV) L1 protein antibodies YFH18L1-A room temperature of 1ng/ml to shake 60 minutes.This after phosphoric acid buffer (pH7.4) cleaning, adds horseradish peroxidase-labeled mountain goat anti-human igg (Zymed company) (1:4000 dilution) for film.Western Blot luminous detection test kit is purchased from PIERCE company.
Detected result as shown in Figure 4, shows the cervical exfoliated cell that anti-human papilloma virus (anti-HPV) L1 protein antibodies YFH18L1-A can specific detection be infected by HPV.
Claims (10)
1. an anti-human papilloma virus (anti-HPV) L1 protein antibodies, comprises variable region of heavy chain and variable region of light chain, it is characterized in that, three hypervariable region CDRH1, CDRH2 of described variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively:
gDSVSSNSNTA,
yYRSKWY,
iIAPDAFDI;three hypervariable region CDRL1, CDRL2 of described variable region of light chain, the aminoacid sequence of CDRL3 are respectively
rASQSISGWLA,
gASTLES,
qQYNNYPWT.
2. anti-human papilloma virus (anti-HPV) L1 protein antibodies as claimed in claim 1, is characterized in that, the aminoacid sequence of described variable region of heavy chain is as shown in SEQ ID No.1, and the aminoacid sequence of variable region of light chain is as shown in SEQ ID No.2.
3. anti-human papilloma virus (anti-HPV) L1 protein antibodies as claimed in claim 1, is characterized in that, described anti-human papilloma virus (anti-HPV) L1 protein antibodies is the antigen-binding portion thereof of whole antibody or whole antibody.
4. anti-human papilloma virus (anti-HPV) L1 protein antibodies as claimed in claim 3, is characterized in that, described whole antibody is IgG1 type.
5. anti-human papilloma virus (anti-HPV) L1 protein antibodies as claimed in claim 3, is characterized in that, described antigen-binding portion thereof is Fab fragment, Fab ' fragment, F (ab ')
2fragment or single-chain antibody.
Coding anti-human papilloma virus (anti-HPV) L1 protein antibodies as claimed in claim 2 gene, it is characterized in that, the nucleotide sequence of encoding heavy chain variable region gene is as shown in SEQ ID No.3, and the nucleotide sequence of encoded light chain variable region gene is as shown in SEQ ID No.4.
7. the recombinant vectors that contains gene as claimed in claim 6.
8. the expression system that contains gene as claimed in claim 6.
9. the application of the anti-human papilloma virus (anti-HPV) L1 protein antibodies as described in as arbitrary in claim 1～5 in the medicine of preparation prevention, treatment human papillomavirus relative disease.
10. the anti-human papilloma virus (anti-HPV) L1 protein antibodies as described in as arbitrary in claim 1～5 detects the application in the reagent of human papillomavirus relative disease in preparation.
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| CN109384848A (en) * | 2017-08-10 | 2019-02-26 | 深圳市雅臣智能生物工程有限公司 | Double targeting antibodies of anti-human papilloma virus (anti-HPV) and anti-CD humanization and combinations thereof, preparation method and application |
| CN112409478A (en) * | 2020-11-24 | 2021-02-26 | 华科同济干细胞基因工程有限公司 | Broad-spectrum HPV antibody and preparation method and application thereof |
| CN116162155A (en) * | 2022-11-15 | 2023-05-26 | 北京义翘神州科技股份有限公司 | anti-HPV 6L1 protein antibody and detection method using same |
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| CN112409478A (en) * | 2020-11-24 | 2021-02-26 | 华科同济干细胞基因工程有限公司 | Broad-spectrum HPV antibody and preparation method and application thereof |
| CN116162155A (en) * | 2022-11-15 | 2023-05-26 | 北京义翘神州科技股份有限公司 | anti-HPV 6L1 protein antibody and detection method using same |
| CN116162155B (en) * | 2022-11-15 | 2023-09-29 | 北京义翘神州科技股份有限公司 | anti-HPV 6L1 protein antibody and detection method using same |
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