WO2015096269A1 - 抗人乳头瘤病毒l1蛋白抗体及其编码基因和应用 - Google Patents
抗人乳头瘤病毒l1蛋白抗体及其编码基因和应用 Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
Definitions
- the invention belongs to the field of genetic engineering technology, and particularly relates to an anti-human papillomavirus L1 protein antibody and a coding gene thereof and application thereof.
- HPV Human Papillomavirus
- L1 major capsid protein
- L2 minor capsid protein
- the genome of HPV contains approximately 8000 base pairs.
- the HPV genome consists of eight early Open Reading Frames (ORFs) (ie El-E8), two late reading frames (ORFs) and one non-coding long-length control region.
- ORFs early Open Reading Frames
- the E6 and E7 genes are most important for cell growth stimulation.
- Studies have found that certain HPV-type E6, E7-encoded E6 and E7 proteins bind to the tumor suppressor genes p53 and Rb, respectively, causing the proliferation of epithelial cells (such as cervical epithelial cells) to be out of control.
- the late reading frame L1 and L2 genes encode the major capsid protein and the minor capsid protein of HPV, respectively, and assemble into the capsid of HPV.
- HPV human papilloma
- Low-risk HPV includes HPV6, 11, 42, 43, 44, etc., which do not cause clinical symptoms or cause benign tumors and tendons in humans, such as human vulgaris, genital warts, which grow on the skin and mucous membranes near the reproductive organs. Papilloma growing on mucous membranes, etc.; and high-risk HPV including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, etc., and infected High epithelial cell lesions including tumor hair There is a high correlation, especially HPV16 and 18.
- HPV findings are associated with certain oral and throat cancers.
- HPV infection of the reproductive tract is a long-term process that can be lurking in cells for several years. Once the opportunity is mature (as the host's immunity is reduced:), the latent virus can resume activity.
- the HPV infection process is usually divided into a latent infection period, a subclinical infection period, a clinical symptom period, and an HPV-related tumor stage. Cervical cancer also has a series of prodromal lesions, namely atypical hyperplasia of the cervical epithelium.
- cervical intraepithelial neoplasia In the pathology, cervical intraepithelial neoplasia (CIN) is usually classified into three grades according to severity: mild intratumoral neoplasia (CIN I), cervical intraepithelial neoplasia (CIN ⁇ ) and cervical intraepithelial neoplasia (CIN III), these precancerous lesions may develop into cervical invasive cancer.
- CIN I mild intratumoral neoplasia
- CIN ⁇ cervical intraepithelial neoplasia
- CIN III cervical intraepithelial neoplasia
- HPV infection process has its own particularity.
- the reproduction of HPV virus is often limited to squamous epithelial cells that are terminally differentiated.
- HPV virus can delay the body's immune response for a long time (Schill JT, Day PM, Kines R. (2010) Current understanding of The mechanism of HPV infection. Gynecol. Oncol. 118:S12-S17.).
- Kimbauer et al. Karlinbauer R, Booy F, Cheng N, Lowy DR, Schiller JT. (1992)
- Papillomavirus LI major capsid protein self-assembles into virus-like particles that are highly immunogenic.
- VLP non-infectious empty-shell virus-like particle
- This VLP structure is similar to natural viral particles and has the same antigenic spatial epitope as the intact virus, which can infect animal models or humans to produce high titers of neutralizing antibodies to protect the body from infection.
- L1 capsid protein provides a good means of developing a prophylactic HPV vaccine (Suzich JA, Ghim SJ, Palmer-Hill FJ, White WI, Tamura JK, Bell JA, et al.
- VLP-induced antibodies can effectively prevent papillomavirus infection [Breitburd FKR, Hubbert NL, Nonnenmacher B, Trin-Dinh-Desmarquet C, Orth G,
- the present invention provides an anti-human papillomavirus LI protein antibody which is a fully human anti-human papillomavirus L1 protein antibody and has high affinity and specificity with human papillomavirus L1 protein (main capsid protein:). it is good.
- An anti-human papillomavirus L1 protein antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the three hypervariable regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are: GGSIRSGDY, SYSGT, TVDSGYDFIPDWFHP, the amino acid sequences of the three hypervariable regions CDRL1, CDRL2, CDRL3 of the light chain variable region are SGSNSNIGNSYVH, RN ORPS, AAWADSLGTYV;
- amino acid sequences of the three hypervariable regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are: GFSLTTTGM, DWDDD, IHRREQGRHWDFDY, three hypervariable regions of the light chain variable region, CDRL1, CDRL2, CDRL3
- the amino acid sequences are SGSSSNLGSNFVY, SNVORPS, AAWDDSLDVLV:
- amino acid sequences of the three hypervariable regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are: GDSVSSNSNTA, YYRSKWY, IIAPDAFDI, three hypervariable regions of the light chain variable region, CDRL1, CDRL2, CDRL3
- the amino acid sequences are RASOSISGWLA, GASTLES, OOYNNYPWTo
- the anti-human papillomavirus L1 protein antibody of the invention substantially protects three technical schemes, namely three anti-human papillomavirus L1 protein antibodies of different heavy chain variable regions and light chain variable regions, for the convenience of distinguishing descriptions, respectively Named as antibody H16L1-A, antibody H16L1-B and antibody H18L1-A.
- the CDRH1 of the antibody H16L1-A is located at positions 26-34 of the heavy chain variable region, the amino acid sequence is shown as SEQ ID N0.5; the CDRH2 is located at positions 54-58 of the heavy chain variable region, and the amino acid sequence is SEQ ID N0.6 As shown, CDRH3 is located at positions 100-114 of the heavy chain variable region; the amino acid sequence is set forth in SEQ ID N0.7.
- the CDRL1 of the antibody H16L1-A is located at positions 23 to 35 of the light chain variable region, the amino acid sequence is shown as SEQ ID N0.8; the CDRL2 is located at positions 51 to 57 of the light chain variable region, and the amino acid sequence is SEQ ID N0.9. Shown; CDRL3 is located at positions 90-100 of the light chain variable region, and the amino acid sequence is shown in SEQ ID NO.
- the CDRH1 of the antibody H16L1-B is located at positions 26-34 of the heavy chain variable region, the amino acid sequence is shown in SEQ ID N0.51, and the CDRH2 is located at positions 54-58 of the heavy chain variable region. As shown in SEQ ID NO: 0.5, CDRH3 is located at positions 100-113 of the heavy chain variable region; the amino acid sequence is set forth in SEQ ID NO.
- the CDRL1 of the antibody H16L1-B is located at positions 23-35 of the light chain variable region, the amino acid sequence is shown as SEQ ID N0.54; the CDRL2 is located at positions 51-57 of the light chain variable region, and the amino acid sequence is SEQ ID N0.55. Shown; CDRL3 is located at positions 90-100 of the light chain variable region, and the amino acid sequence is shown in SEQ ID N 0.56.
- the CDRH1 of the antibody H18L1-A is located at positions 26-36 of the heavy chain variable region, the amino acid sequence is shown as SEQ ID N0.61; the CDRH2 is located at positions 56-62 of the heavy chain variable region, and the amino acid sequence is SEQ ID N0.62 As shown, CDRH3 is located at positions 104-112 of the heavy chain variable region; the amino acid sequence is set forth in SEQ ID NO.
- the CDRL1 of the antibody H18L1-A is located at positions 24-34 of the light chain variable region, the amino acid sequence is shown as SEQ ID N0.64; the CDRL2 is located at positions 50-56 of the light chain variable region, and the amino acid sequence is SEQ ID N0.65 Shown; CDRL3 is located at positions 89-97 of the light chain variable region, and the amino acid sequence is shown in SEQ ID N0.66.
- the amino acid sequence of the three hypervariable regions has a large variability, while the amino acid sequence of the region between the hypervariable regions changes little.
- These three hypervariable regions are spatially structurally complementary to antigenic determinants and are therefore referred to as complementary determinants (CDRs).
- CDRs of the different heavy and light chains determine the specificity of the antibody for the antigen.
- amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 2 (antibody H16L1-A);
- amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 47, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 48 (antibody H16L1-B);
- the anti-human papillomavirus L1 protein antibody may be an antigen-binding portion of a whole antibody or a whole antibody.
- the whole antibody is preferably of the IgG1 type; the antigen binding portion is preferably a Fab fragment, a Fab' fragment, a F (ab':> 2 fragment or a single chain antibody, more preferably a single chain antibody.
- the antigen-binding portion retains both a region that specifically binds to the antigen and a side effect caused by the antigenicity of the Fc fragment.
- the single-chain antibody has the advantages of increasing the drug concentration in the easily permeable tumor tissue, small immunogenicity, short half-life in the circulation in the body, easy to remove, easy to be linked with the toxin or the enzyme gene, and directly obtaining the immunotoxin or the enzyme-labeled antibody.
- the antigen binding portion can be prepared by recombinant DNA techniques or by enzymatic/chemical cleavage of whole antibodies.
- the preparation method of the anti-human papillomavirus L1 protein single-chain antibody of the present invention is: constructing a human single-chain antibody library by the method disclosed in Chinese Patent Publication No. CN 1444651 A, and screening the human single-chain antibody library for anti-human Papillomavirus L1 protein antibody.
- the present invention also provides a gene encoding the anti-human papillomavirus L1 protein antibody, wherein the nucleotide sequence encoding the heavy chain variable region gene is represented by SEQ ID No. 3, encoding a light chain variable region gene
- SEQ ID No. 4 antibody H16L1-A
- nucleotide sequence encoding the heavy chain variable region gene is represented by SEQ ID No. 49
- nucleotide sequence encoding the light chain variable region gene is represented by SEQ ID No. 50 (antibody H16L1-B);
- nucleotide sequence encoding the heavy chain variable region gene is represented by SEQ ID No. 59
- nucleotide sequence encoding the light chain variable region gene is represented by SEQ ID No. 60 (antibody H18L1-A).
- the invention also provides a recombinant vector or expression system comprising the encoding gene.
- the original vector of the recombinant vector is pACT2 or pET27b.
- the present invention also provides the use of the anti-human papillomavirus L1 protein antibody for the preparation of a medicament for preventing or treating a human papillomavirus-associated disease.
- the present invention also provides the use of the anti-human papillomavirus L1 protein antibody for the preparation of a reagent for detecting a disease associated with human papillomavirus.
- the anti-human papillomavirus L1 protein antibody of the present invention specifically binds to human papillomavirus L1 protein and detects HPV-infected cells.
- the HPV-infected cells can be detected by using the full-length recombinant human anti-human papillomavirus L1 protein antibody, and therefore, the anti-human papillomavirus L1 protein antibody of the present invention can be used to prepare a reagent for detecting human papillomavirus-related diseases. .
- the human papillomavirus-related disease may be any disease (directly or indirectly caused) associated with human papillomavirus (HPV) infection, such as cervical cancer, anal cancer, vaginal cancer, Penile cancer, oral cancer, throat cancer, etc.
- HPV human papillomavirus
- the human papillomavirus-related disease is specifically a cervical intraepithelial neoplasia or cervical cancer caused by human papillomavirus
- the human papillomavirus-related disease may be associated with human papillomavirus type 16 infection.
- Disease antibody H16L1-A or H16L1-B can be applied at this time
- human papillomavirus type 18 Infection-related diseases antibody H18L1-A can be applied at this time).
- the anti-human papillomavirus L1 protein antibody of the invention is a full-human anti-human papillomavirus L1 protein antibody which can be used for detecting HPV infection, and can also be used for preventing and treating human diseases, has high affinity and can specifically bind human papilloma.
- the viral L1 protein therefore, can be formulated into a drug or agent to effectively detect, prevent, and treat diseases caused by HPV infection.
- Figure 1 is a schematic view showing the structure of a single-chain antibody of the present invention.
- Figure 2 shows the results of Coomassie blue staining of human papillomavirus type 16 L1 protein expressed in insect s ⁇ cells after SDS-PAGE.
- the first lane s ⁇ total cell protein after empty vector infection
- lane 2 total protein of sf9 cells after infection with HPV16-L1-containing vector
- lane 3 purified HPV16-type L1 protein.
- Figure 3 is a ELISA test result of the anti-human papillomavirus L1 protein single chain antibody #H16L1-A of the present invention.
- Figure 4 is a Western Blot of the anti-human papillomavirus L1 protein antibody YFH16L1-A; wherein, lane 1: normal cervical cell sample; lane 2: cervical exfoliated cells of CIN stage 2; lane 3: CIN3 phase Cervical exfoliated cells of the patient; Lane 4: Cervical exfoliated cells of patients with cervical cancer.
- Fig. 5 is a ELISA test result of the anti-human papillomavirus L1 protein single chain antibody #H16L1-B of the present invention.
- Figure 6 shows the results of ELISA assay of the anti-human papillomavirus L1 protein single chain antibody #H18L1-A of the present invention.
- Figure 7 is a Western Blot of the anti-human papillomavirus L1 protein antibody YFH16L1-B of the present invention.
- lane 1 normal cervical cell sample
- lane 2 cervical exfoliated cells of CIN stage 2 patient
- lane 3 CIN3 phase Cervical exfoliated cells of the patient
- Lane 4 Cervical exfoliated cells of patients with cervical cancer.
- Figure 8 is a result of Coomassie blue staining of human papillomavirus type 18 L1 protein expressed by insect s ⁇ cells in SDS-PAGE of the present invention; wherein, the first lane: s ⁇ total cell protein after empty vector infection; : Total protein of sf9 cells after infection with HPV18-L1-containing vector; Road: Purified HPV18 type LI protein.
- Figure 9 is a Western Blot of the anti-human papillomavirus L1 protein antibody YFH18L1-A of the present invention. wherein, lane 1: normal cervical cell sample; lane 2: cervical exfoliated cells of CIN stage 2 patient; lane 3: cervical cancer The patient's cervical exfoliated cells.
- Example 1 Screening of single-chain antibody against human papillomavirus L1 protein
- the human single-chain antibody library was constructed by the method disclosed in Chinese Patent Publication No. CN 1444651 A, and the human papillomavirus L1 protein single-chain antibody was screened in the human single-chain antibody library, and the specific implementation process was as follows:
- Poly A+ RNA (purchased from Clontech) from human bone marrow, human fetal liver, human spleen and human peripheral blood leukocytes was used as a template, and oligo (dT) and random primers were used, and a reverse transcriptase kit was used. (purchased from Clontech), reverse transcription of poly A+ RNA into cDNA according to the method guidelines provided by the Clontech kit.
- VH heavy chain variable region
- VL light chain variable region
- the first set is a 5'-end primer (SEQ ID No. ll-17) that amplifies the human antibody heavy chain variable region (VH) gene, including:
- VHlb 5 - CCATACGATGTTCCAGATTACCAGGTGCAGCTGCAG
- VH2b 5 -CCATACGATGTTCCAGATTACCAGGTACAGCTGCAGC
- VH3b 5,-CCATACGATGTTCCAGATTACCAGGTGCAGCTACAGCA
- VH4b ;, -CCATACGATGTTCCAGATTACGAGGTGCAGCTG(G/TT GGAG(A/T)C(C/T)-3,;
- VH7b ;, -CCATACGATGTTCCAGATTACCAGGTGCAGCTGGTG
- the second set is a 3'-end primer (SEQ ID No. 18-23) that amplifies the human antibody heavy chain variable region (VH) gene, including:
- VHlf 5,-GCCGCCTGATCCACCACCACCGCCTGAGGAGAC(A/G)GTGA
- VH2f ;, -GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACC
- VH3f i'-GCCGCCTGATCCACCACCGCCTGAAGAGACGGTGACC
- VH4f i'-GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACC
- VH5f ;, -GCCGCCTGATCCACCACCGCCGGTTGGGGCGGATGC
- VH6f 5,-GCCGCCTGATCCACCACCGCC(C/G)GATGGGCCCTTGG
- the third group is a 5,-terminal primer (SEQ ID No. 24-32) which amplifies the human antibody ⁇ -light chain variable region ( ⁇ ) gene, and includes:
- VL9b ;, -GGCAGCGGTGGTGGAGGCAGTAATTTTATGCTGACTCA
- the fourth group is a 3'-end primer (SEQ ID No. 33-34) which amplifies the human antibody ⁇ -light chain variable region ( ⁇ ) gene, and includes:
- VL2f 5, -GGGGTTTTTCAGTATCTACGAGAGGACGGTCAGCTGG
- the fifth group is a 5'-end primer (SEQ ID No. 35-38) for amplifying the human antibody k-light chain variable region (Vk) gene, including:
- VK4b ;, -GGCAGCGGTGGTGGAGGCAGTGAAACGACACTCACG
- the sixth group is a 3'-end primer (SEQ ID No. 39-42) for amplifying the human antibody k-light chain variable region (Vk) gene, including: VKlf: 5'-GGGGTTTTTCAGTATCTACGATTTGATTTCCACCTTGG
- VK3f -GGGGTTTTTCAGTATCTACGATTTGATATCCACTTTGG
- VK4f -GGGGTTTTTCAGTATCTACGATTTAATCTCCAGTCGTG
- the first set of primers contained the yeast two-hybrid vector 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.
- Upstream homologous sequence of the multiple cloning site (underlined portion); yeast and two-hybrid vectors in the fourth and sixth primers
- the downstream homologous sequence of the pACT2 multiple cloning site (underlined); while the second, third, and fifth primers contain a linker peptide sequence (underlined), and the linker peptide is used to link the heavy chain of the antibody. Zone and light chain variable regions.
- the PCR reaction system When amplifying, the PCR reaction system and the reaction conditions are the same, and the PCR reaction system is:
- Taq polymerase (Takara) 1.0 unit water (ddH 2 0 ) 36 ⁇
- the above components were uniformly mixed and placed in a PCR machine to carry out a reaction.
- the reaction conditions were: melting at 94 °C for 1 minute, annealing at 50 °C for 1 minute, extension at 72 °C for 2.5 minutes, and cycling 30 times.
- the upstream primer (primer 7, SEQ ID No. 43, underlined is the upstream homologous sequence of the vector pACT2 multiple cloning site):
- Downstream primers (primer 8, SEQ ID No. 44, underlined for the linker peptide reverse strand sequence): 5 '-ACTGCCTCCACCACCGCT
- PCR amplification was carried out, and the reaction conditions and system were the same as in step 1. Upon completion of the reaction, a human antibody heavy chain variable region DNA sequence containing the upstream homologous sequence of the 5'-carrier pACT2 multiple cloning site and the 3'-linked peptide reverse strand sequence was obtained.
- the human antibody light chain variable region DNA obtained by PCR in the step 1 is used as a template, and the primer 9 and the primer 10 are respectively a downstream primer and an upstream primer, and the sequence is as follows:
- Upstream primer (primer 10, SEQ ID No. 45, underlined part is the linker peptide sequence): 5'-GGCGGTGGTGGATCAGGCGGCGGAGGATCTGGCGGAGGTGG
- the downstream primer (primer 9, SEQ ID No. 46, underlined is the downstream homologous sequence of the vector pACT2 multiple cloning site):
- PCR amplification was carried out, and the reaction conditions and system were the same as in step 1. Upon completion of the reaction, a human antibody light chain variable region DNA sequence comprising a downstream homologous sequence of the 3'-carrier pACT2 multiple cloning site and a 5'-linked peptide cis sequence was obtained.
- scFv single-chain antibody
- the single-chain antibody (scFv) DNA obtained in the step 3 and the yeast two-hybrid vector pACT2 treated with restriction enzymes (Mm HI and Eco RI) were used according to the method provided by the original Clontech Co., Ltd.
- yeast Protocol Handbook Co-transplanted into yeast strain ⁇ 187 ( ⁇ % draw 3-52, his3-200, ade2-101, lys2-801, trp 1-901, leu2-3, 112, gaU ⁇ , gal80A, met-, URA3:: GAL1 UAS
- the single-chain antibody DNA was integrated into the pACT2 vector after intracellular homologous recombination, thereby obtaining a yeast two-hybrid single-chain antibody library, a single-chain antibody DNA fragment and Gal4 on the pACT2 vector.
- the Activation Domain (AD) is fused together.
- Antibodies DNA copy number yeast two hybrid single chain antibody gene library by homologous recombination obtained about IX 10 8 th, it may be applied to yeast two-hybrid screening of specific antibody.
- Human papillomavirus type 16 (HPV16) L1 protein was used as an antigen to screen the yeast two-hybrid single-chain antibody gene library using human papillomavirus type 16 (HPV16) L1 protein (capsid major protein:) as an antigen. Antibody screening.
- the DNA encoding the human papillomavirus HPV16 type L1 protein was recombined into the vector pGBKT7 to construct pGBK-H16L1.
- pGBK-H16L1 encodes the Gal4 DNA binding region (Binding Domain, BD for short) and fuses human papillomavirus HPV16 L1 protein at its C-terminus.
- the PGBK-H16L1 plasmid DNA was transformed into yeast strain AH109 (MATa, trp 1-901, leu2-3, 112, painting 3-52, his3-200, gal4 ⁇ , gal80A, LYS2: : GAL1 GAL1 dish-HIS3, GAL2u AS - GAL2 TATA -ADE2, URA3:: MEL1 -MELln-lac Z ); with pGBK-H16Ll plasmid
- the AH109 yeast can be grown on a tryptophan-free synthetic medium (SD/-W).
- yeast cells containing the two plasmids can be grown in yeast synthesis medium containing no leucine and serine ( SD/-LW).
- Human papillomavirus type 18 (HPV18) L1 protein was used as an antigen to screen the yeast two-hybrid single-chain antibody gene library using human papillomavirus type 18 (HPV18) L1 protein (capsid major protein:) as an antigen. Antibody screening.
- the DNA encoding the human papillomavirus HPV18 type L1 protein was recombined into the vector pGBKT7 to construct pGBK-H18L1.
- pGBK-H18L1 encodes a Gal4 DNA binding region (Binding Domain, BD for short) and fuses human papillomavirus HPV18 L1 protein at its C-terminus.
- the PGBK-H18L1 plasmid DNA was transformed into yeast strain AH109 (MATa, trpl-901, leu2-3, 112, drawing 3-52, his3-200, Gal4 A, gal80A, LYS2: : GAL1 ⁇ -GAL1 TATA -H1S3, GAL2u A& - GAL2 TATA -ADE2, URA3:: MEL1 -MELln-lac Z ); AH109 yeast with pGBK-H18Ll plasmid can be free of color ammonia Growth on acid synthetic medium (SD/-W).
- yeast strain AH109 MATa, trpl-901, leu2-3, 112, drawing 3-52, his3-200, Gal4 A, gal80A, LYS2: : GAL1 ⁇ -GAL1 TATA -H1S3, GAL2u A& - GAL2 TATA -ADE2, URA3:: MEL1 -MELln-lac Z ); AH
- MA-type yeast cells AH109 strain
- PGBK-H18L1 PGBK-H18L1
- Y187 strain yeast-type yeast cell
- yeast containing both plasmids The cells can be grown in yeast synthetic medium (SD/-LW) without leucine and serine.
- the detection of the expression of ⁇ -galactosidase in the yeast cell can determine the presence of the scFv/L1 protein in the yeast cell.
- the pACT2 plasmid DNA containing scFv was extracted from the above 37 ⁇ -galactosidase-positive yeasts, and then combined with pGBKT7 empty vector DNA, pGBKT-IRa plasmid DNA, pGBKT-Lam plasmid DNA (encoding the Gal4 DNA binding region and in its C).
- the human fusion laminin C) is co-transformed into AH109 yeast cells; the transformed yeast cells are first grown on SD/-LW plate medium and then transferred to SD/-AHLW plate medium; The colonies were analyzed for ⁇ -galactosidase, and colonies that were verified to be positive were considered to contain non-specific scFv and were knocked out.
- the DNA sequence of the heavy chain variable region of #H16L1-A is shown in SEQ ID No. 3.
- the amino acid sequence of the #H16L1-A heavy chain variable region (SEQ ID No. 1) is:
- the underlined portion is followed by three hypervariable regions CDRH1, CDRH2, CDRH3 (SEQ ID No. 5-7);
- the DNA sequence of the light chain variable region of #H16L1-A is shown in SEQ ID No. 4.
- the #H16L1-A region amino acid sequence (SEQ ID No. 2) is:
- the underlined part is the three hypervariable regions CDRL1, CDRL2, CDRL3 (SEQ ID) ⁇ 8 ⁇ 10).
- the DNA sequence of the heavy chain variable region of #H16L1-B is shown in SEQ ID No. 49.
- amino acid sequence of the heavy chain variable region of #H16L1-B (SEQ ID No. 47) is:
- the underlined portion is followed by three hypervariable regions CDRH1, CDRH2, CDRH3 (SEQ ID No. 51-53);
- the DNA sequence of the light chain variable region of #H16L1-B is shown in SEQ ID No. 50.
- amino acid sequence of the #H16L1-B region (SEQ ID No. 48) is:
- the underlined portions are three hypervariable regions CDRL1, CDRL2, CDRL3 (SEQ ID ⁇ 54 ⁇ 56).
- the pACT2 plasmid DNA containing scFv was extracted from the above 23 ⁇ -galactosidase-positive yeasts, and then combined with pGBKT7 empty vector DNA, pGBKT-IRa plasmid DNA, pGBKT-Lam plasmid DNA (encoding the Gal4 DNA binding region and in its C).
- the human fusion laminin C) is co-transformed into AH109 yeast cells; the transformed yeast cells are first grown on SD/-LW plate medium and then transferred to SD/-AHLW plate medium; The colonies were analyzed for ⁇ -galactosidase, and colonies that were verified to be positive were considered to contain non-specific scFv and were knocked out.
- the DNA sequence of the heavy chain variable region of #H18L1-A is shown in SEQ ID No. 59.
- amino acid sequence (SEQ ID No. 57) of the heavy chain variable region of #H18L1-A is: AVYYCTKIIAPDAFDIWGQGTMVTVSS;
- the DNA sequence of the light chain variable region of #H18L1-A is shown in SEQ ID No. 60.
- the amino acid sequence of the #H18L1-A region (SEQ ID No. 58) is:
- Example 2 Recombinant Human Papillomavirus HPV16 Type L1 Protein, HPV18 Type L1 Protein Expression and Preparation of Rabbit Antiserum
- HPV16 L1 coding DNA fragment is optimized according to the codon frequency of the s ⁇ insect cell to adjust the codon of the HPV16 type L1 encoding DNA, and the optimized coding DNA is inserted into the pFastBac Dual vector Xba I site to obtain a recombinant vector/ ?FastBac-HPV16Ll o
- Recombinant Bacmido was obtained by transforming E. coli DHlOBac competent cells. Recombinant Bacmid was used to transfect insect s ⁇ cells, and cultured in a constant temperature incubator for 72 hours, the cell culture supernatant was collected to obtain HPV16-type L1 protein. Recombinant baculovirus.
- the recombinant virus infects sf9 cells in large quantities. After 72 hours, the infected cells were collected, sonicated, centrifuged at 12,000 rpm for 10 minutes at 4 °C, and the supernatant was taken. The VLP was purified by CsCl density gradient ultracentrifugation (Shi W, Liu J, Huang). Y et al. (2001) Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses. J Virol 75: 10139-10148.). Finally, dialyzed against dialysate (10 mM HEPES, 150 mM NaCl) for 3 hours to obtain purified HPV type 16 LI protein (see Figure 2).
- Rabbit anti-HPV16 type L1 serum was prepared according to the conventional method of Harlow et al. (Harlow E, Hekou Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, pages 53 to 138).
- the purified HPV 16 L1 protein was diluted to 400 ⁇ l and fully emulsified with an equal volume of Freund's adjuvant.
- the rabbits were injected subcutaneously at 0, 4, 8 and 12 weeks.
- the blood was taken intravenously 1 week after the last immunization, and the serum antibody titer was measured by ELISA. After the titer reached the expected level, blood was taken and serum was collected.
- HPV18 L1 protein expression and rabbit antiserum were prepared in the same manner as HPV16 L1 protein. The results are shown in Fig. 8.
- Example 3 Specificity detection
- the gene encoding the single-chain antibody #H16L1 -A was cloned into the expression vector pET27b(+) to construct pET27b-H16Lla;
- pET27b-H16Lla was transformed into the expression bacterium E. coli BL21 (DE3), and induced by IPTG (0.5 mM) according to the method provided by Novagen; in the expressed target protein, the N-terminus of scFv was pelB sequence, and the pelB sequence was The expressed scFv is secreted into the periplasmic space of BL21 (DE3); the C-terminus of scFv contains an HSV marker and a 6 X His marker to facilitate purification of the target protein;
- the single-chain antibody against human papillomavirus HPV16 type L1 protein was conveniently isolated and purified using a Ni-NTA column using the method provided by Qiagen.
- Single-chain antibody #11161 ⁇ -:6, #H18L1 -A expression and purification method can refer to the single-chain antibody #H16L1-A method, the single-chain antibody #H16L1-A coding gene corresponding to the single-chain antibody# The coding gene of 11161 ⁇ -:6 or #11181 ⁇ - can be used.
- HPV type 16 L1 protein expressed and purified by insect cell sf9 is shown in Example 2 above.
- the ELISA test method is as follows: (1) coating a 96-well plate with HPV type 16 LI protein, overnight at 2-8 ° C;
- the specific detection method of the single-chain antibody #11161 ⁇ -8 is the same as that of the single-chain antibody #11161 ⁇ -, and only the single-chain antibody #H16L1-A can be replaced with the single-chain antibody #11161 ⁇ -:6.
- Example 2 3 ELISA test single-chain antibody #H18L1-A specific for HPV18 type L1 protein Insect cell sf9 expressed and purified HPV18 type L1 protein is shown in Example 2 above.
- the ELISA test method was the same as the above "2 ELISA test for single-chain antibody #H16L1-A P#H16Ll-B for HPV type 16 L1 protein".
- the company was commissioned by Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. to prepare full-length recombinant human anti-human papillomavirus L1 protein antibodies YFH16L1-A, YFH16L1-B and YFH18L1-A.
- YFH16L1-A, YFH16L1-B and YFH18L1-A are all IgGl type
- the variable region sequence of YFH16L1-A is identical to the variable region sequence of single-chain antibody #H16L1-A
- the variable region sequence and single strand of YFH16L1-B The variable region sequences of antibody #11161 ⁇ -8 were identical, and the variable region sequence of YFH18L1-A was identical to the variable region sequence of single-chain antibody #YFH18L1-A.
- cervical intraepithelial neoplasia II ie CIN2
- cervix Cervical exfoliated cells of stage III ie, CIN stage 3
- cervical cancer patients with intraepithelial neoplasia were obtained from the Obstetrics and Gynecology Hospital affiliated to Zhejiang University Medical College and obtained informed consent.
- test results are shown in Fig. 4, Fig. 7, and Fig. 9, indicating that the human papillomavirus L1 protein antibodies YFH16L1-A, YFH16L1-B and YFH18L1-A can specifically detect HPV-infected cervical exfoliated cells.
- Ggggtttttc agtatctacg ataggacggt sascttggtc c
- Ggggtttttc agtatctacg agaggacggt cagctgggtg c
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Abstract
本发明公开了一种抗人乳头瘤病毒L1蛋白抗体及其编码基因和应用,抗人乳头瘤病毒L1蛋白抗体包括重链可变区和轻链可变区,重链可变区的三个高变区的氨基酸序列分别如SEQ ID NO.5~7所示,轻链可变区的三个高变区的氨基酸序列分别分别如SEQ ID NO.8~10所示;或重链可变区的三个高变区的氨基酸序列分别如SEQ ID NO.51~53所示,轻链可变区的三个高变区的氨基酸序列分别如SEQ ID NO.54~56所示;或重链可变区的三个高变区的氨基酸序列分别如SEQ ID NO.61~63所示,轻链可变区的三个高变区的氨基酸序列分别如SEQ ID NO.64~66所示。本发明抗体与人乳头瘤病毒L1蛋白亲和力高。
Description
抗人乳头瘤病毒 L1蛋白抗体及其编码基因和应用 技术领域
本发明属于基因工程技术领域, 尤其涉及一种抗人乳头瘤病毒 L1蛋 白抗体及其编码基因和应用。
说
背景技术
人乳头瘤病毒(Human Papillomavirus, 缩写 HPV)是一种具有种属特 异性的嗜上皮病毒, 属双链闭环的小 DNA病毒。 HPV病毒由蛋白衣壳这 层外衣和被包裹着的双链环状的 DNA核心构成。 蛋白衣壳由主要衣壳蛋 白 (L1 ) 和次要衣壳蛋白 (L2) 组成。 1949年, Strauss首先在电镜下观 察到 HPV颗粒, 它呈球形, 其 20面体对称, 直径书约 45-55nm。
HPV的基因组包含约 8000个碱基对。 HPV基因组包括 8个早期开放读 码框架 (Open Reading Frame, 简称 ORF) (即 El -- E8 )、 2个晚期读码框 架 (ORF) 和 1个非编码长控区。 在早期开放读码框架中, E6和 E7基因对 细胞生长刺激最为重要。 研究发现某些 HPV型别 E6、 E7编码的 E6、 E7蛋 白分别与抑癌基因 p53和 Rb结合, 引起上皮细胞 (如宫颈上皮细胞) 增殖 失控。而晚期读码框 L1和 L2基因分别编码 HPV的主要衣壳蛋白和次要衣壳 蛋白, 组装成 HPV的衣壳。
根据病毒分析已经发现 100余种类型的人乳头状瘤病。 目前基因组序 列已经确定的 HPV型别约有 80余种, 依其感染的上皮所在部位分为皮肤 型 HPV和生殖道上皮 HPV, 大约 35种型别可感染妇女生殖道, 约 20种 与肿瘤相关。 依据不同型别 HPV与肿瘤发生的危险性高低分为低危险型 别和高危险型别 HPV。 低危险型别 HPV包括 HPV6、 11、 42、 43、 44等 型别, 不引起临床症状或引起人类良性的肿瘤和疣, 如生长在生殖器官附 近皮肤和粘膜上的人类寻常疣、 尖锐湿疣以及生长在粘膜上的乳头状瘤 等; 而高危险型 HPV包括 HPV16、 18、 31、 33、 35、 39、 45、 51、 52、 56、 58、 59、 66、 68等型别, 与被感染的上皮细胞高度病变包括肿瘤的发
生有很高的相关性, 尤其是 HPV16和 18型。 这些类型的人乳头状瘤病毒 主要与生殖道恶性病变的发生有关, 如子宫颈癌、 肛门癌、 阴道癌和阴茎 癌。 此外, 高危型 HPV发现与某些口腔和咽喉癌有关。
HPV感染生殖道是一个长期的过程, 可潜伏在细胞内若干年, 一旦机 会成熟 (如宿主机体免疫力降低:), 潜伏的病毒可恢复活动。 HPV感染过程 通常分为潜伏感染期、 亚临床感染期、 临床症状期和 HPV相关的肿瘤期。 宫颈癌也有一系列的前驱病变, 即宫颈上皮不典型增生, 在病理上称宫颈 上皮内瘤变 ( cervical intraepithelial neoplasia, 缩写 CIN), 通常又根据严 重程度分成三级:宫颈上皮内轻度瘤变( CIN I )、宫颈上皮内中度瘤变( CIN Π )和宫颈上皮内高度瘤变(CIN III ) , 这些癌前病变均有可能发展为宫颈 浸润癌。
HPV的感染过程有其特殊性。 HPV病毒的繁殖往往仅限于处于末端分 化的鳞状上皮细胞内, 此外, HPV病毒能在相当长时间内延迟人体对其产 生免疫反应 (Schill JT, Day PM, Kines R. (2010) Current understanding of the mechanism of HPV infection. Gynecol. Oncol. 118:S12-S17.)。 近年来 HPV感 染过程研究的长足发展得益于现代分子生物学的进展。 Kimbauer等证明 (Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT. (1992) Papillomavirus LI major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci USA. 89: 12180— 12184.)主要衣壳蛋白 (LI 蛋白)单独表达或者与次要衣壳蛋白 (L2蛋白)共表达均可自组装成无感 染性的空衣壳病毒样颗粒 (virus-like-particle, 简称 VLP), 并具有很高的 免疫原性。 VLP可以有效地通过多种上皮细胞和培养的细胞系表面的受体 与细胞相结合 [Roden RB, Kirnbauer R, Jenson AB, Lowy DR, Schiller JT.
(1994) Interaction of papillomaviruses with the cell surface. J Virol. 68:
7260-7266.]。 这种 VLP结构与天然的病毒颗粒相似, 具有与完整病毒相同 的抗原空间表位, 可感染诱导动物模型或人体产生高滴度的中和抗体, 以 保护机体免受感染。
L1衣壳蛋白的这一特性为开发预防性 HPV疫苗提供了良好的手段 (Suzich JA, Ghim SJ, Palmer-Hill FJ, White WI, Tamura JK, Bell JA, et al.
(1995) Systemic immunization with papillomavirus LI protein 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 type 4 in prophylactic and therapeutic immunization. Virology 219:3744; 禾卩 Schiller JT. Lowy DL. (2006) Prospects for cervical cancer prevention by human
papillomavirus vaccination. Cancer Res. 66: 10229—10232)。
VLP诱导产生的抗体可以有效地预防乳头瘤病毒的感染 [Breitburd FKR, 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; 和 Suzich JA, Ghim SJ, Palmer-Hill FJ, White WI, Tamura JK, Bell JA, et al. (1995) Systemic immunization with papillomavirus LI protein completely prevents the development of viral mucosal papillomas. Proc Natl Acad Sci USA. 92: 11553-11557]。 研究表明, 抗 VLP的单克隆抗体也能有效地中和 HPV病毒对人细胞的感染性 (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·)。
此外,有研究表明检测 HPV LI蛋白在宫颈人乳头状瘤病毒感染的诊断 和预后判定中的也有作用 (贾咏存,樊杨, 纳文霞(2010 ) .HPV L1检测在宫 颈人乳头状瘤病毒感染预后判定中的作用。《宁夏医学杂志》 8 : 688-690]; [齐瑞玲,贾晓云,唐思源 (2012) 人乳头状瘤病毒 L1壳蛋白检测对宫颈疾 病诊断价值. 《中华实用诊断与治疗杂志》 26(8):785-786] ; [史健, 邓宽 国, 刘玉霞, 尹石华 (2010) 人乳头瘤病毒及其 L1蛋白检测在宫颈病变诊 断中的应用。 医学检验与临床. 21(6):70-76]。
因此, 获得能检测与中和人乳头瘤病毒的全人源抗 L1衣壳蛋白抗体 对于检测和治疗 HPV引起的疾病显得非常有意义。 发明内容
本发明提供了一种抗人乳头瘤病毒 LI蛋白抗体, 该抗体为全人源抗 人乳头瘤病毒 L1蛋白抗体, 与人乳头瘤病毒 L1蛋白 (衣主要壳蛋白:)具有 高亲和力, 特异性好。
一种抗人乳头瘤病毒 L1蛋白抗体, 包括重链可变区和轻链可变区, 所述重链可变区的三个高变区 CDRH1、 CDRH2、 CDRH3 的氨基酸序列 分别为: GGSIRSGDY、 SYSGT、 TVDSGYDFIPDWFHP, 所述轻链可变 区的三个高变区 CDRL1、 CDRL2、 CDRL3 的氨基酸序列分别为 SGSNSNIGNSYVH, RN ORPS, AAWADSLGTYV;
或所述重链可变区的三个高变区 CDRH1、 CDRH2、 CDRH3 的氨基 酸序列分别为: GFSLTTTGM、 DWDDD、 IHRREQGRHWDFDY, 所述轻 链可变区的三个高变区 CDRL1、 CDRL2、 CDRL3 的氨基酸序列分别为 SGSSSNLGSNFVY, SNVORPS, AAWDDSLDVLV:
或所述重链可变区的三个高变区 CDRH1、 CDRH2、 CDRH3 的氨基 酸序列分别为: GDSVSSNSNTA, YYRSKWY, IIAPDAFDI, 所述轻链可 变区的三个高变区 CDRL1、 CDRL2、 CDRL3 的氨基酸序列分别为 RASOSISGWLA, GASTLES, OOYNNYPWTo
本发明的抗人乳头瘤病毒 L1蛋白抗体实质上保护三种技术方案, 即 三种不同重链可变区和轻链可变区的抗人乳头瘤病毒 L1蛋白抗体, 为便 于区分描述, 分别依次命名为抗体 H16L1-A、 抗体 H16L1-B 和抗体 H18L1-A。
抗体 H16L1-A的 CDRH1位于重链可变区第 26~34位,氨基酸序列如 SEQ ID N0.5所示; CDRH2位于重链可变区第 54~58位, 氨基酸序列如 SEQ ID N0.6所示, CDRH3位于重链可变区第 100~114位; 氨基酸序列 如 SEQ ID N0.7所示。
抗体 H16L1-A的 CDRL1位于轻链可变区第 23~35位,氨基酸序列如 SEQ ID N0.8所示; CDRL2位于轻链可变区第 51~57位, 氨基酸序列如 SEQ ID N0.9所示; CDRL3位于轻链可变区第 90~100位, 氨基酸序列如 SEQ ID NO.10所示。
抗体 H16L1-B的 CDRH1位于重链可变区第 26~34位,氨基酸序列如 SEQ ID N0.51所示; CDRH2位于重链可变区第 54~58位, 氨基酸序列如
SEQ ID N0.52所示, CDRH3位于重链可变区第 100~113位; 氨基酸序列 如 SEQ ID NO.53所示。
抗体 H16L1-B的 CDRL1位于轻链可变区第 23~35位,氨基酸序列如 SEQ ID N0.54所示; CDRL2位于轻链可变区第 51~57位, 氨基酸序列如 SEQ ID N0.55所示; CDRL3位于轻链可变区第 90~100位, 氨基酸序列 如 SEQ ID N0.56所示。
抗体 H18L1-A的 CDRH1位于重链可变区第 26~36位,氨基酸序列如 SEQ ID N0.61所示; CDRH2位于重链可变区第 56~62位, 氨基酸序列如 SEQ ID N0.62所示, CDRH3位于重链可变区第 104~112位; 氨基酸序列 如 SEQ ID NO.63所示。
抗体 H18L1-A的 CDRL1位于轻链可变区第 24~34位,氨基酸序列如 SEQ ID N0.64所示; CDRL2位于轻链可变区第 50~56位, 氨基酸序列如 SEQ ID N0.65所示; CDRL3位于轻链可变区第 89~97位, 氨基酸序列如 SEQ ID N0.66所示。 在抗体分子可变区上, 三个高变区的氨基酸序列存 在很大的变异性, 而高变区之间的区域氨基酸序列则变化较小。 这三个高 变区在空间结构上可与抗原决定簇形成精密的互补, 因此又被称为互补决 定区 (CDR), 不同重链、 轻链的 CDR决定了抗体对抗原的特异性。
优选的, 所述重链可变区的氨基酸序列如 SEQ ID No.l所示, 所述轻 链可变区的氨基酸序列如 SEQ ID No.2所示 (抗体 H16L1-A);
或所述重链可变区的氨基酸序列如 SEQ ID No.47所示, 所述轻链可 变区的氨基酸序列如 SEQ ID No.48所示 (抗体 H16L1-B);
或所述重链可变区的氨基酸序列如 SEQ ID No.57所示, 所述轻链可 变区的氨基酸序列如 SEQ ID No.58所示(抗体 H18L1-A)。所述抗人乳头 瘤病毒 L1蛋白抗体可以为全抗体或全抗体的抗原结合部分。 所述的全抗 体优选为 IgGl型;所述的抗原结合部分优选为 Fab片段、 Fab'片段、 F(ab':>2 片段或单链抗体, 更优选为单链抗体。
抗原结合部分既保留了能与抗原特异结合的区域, 又避免了 Fc片段 的抗原性而引起的副作用。 其中, 单链抗体具有易渗透肿瘤组织中增加药 物浓度, 免疫原性小, 在体内循环的半衰期短、 易于清除, 易于与毒素或 酶基因连接从而直接获得免疫毒素或酶标抗体等优点。
抗原结合部分可以通过 DNA重组技术或通过酶促 /化学裂解全抗体来 制备。 本发明抗人乳头瘤病毒 L1蛋白单链抗体的制备方法为: 采用公开 号为 CN 1444651 A的中国专利文献公开的方法构建人单链抗体文库, 并 在该人单链抗体文库中筛选抗人乳头瘤病毒 L1蛋白抗体。
本发明还提供了编码所述的抗人乳头瘤病毒 L1蛋白抗体的基因, 其 中, 编码重链可变区基因的核苷酸序列如 SEQ ID No.3所示, 编码轻链可 变区基因的核苷酸序列如 SEQ ID No.4所示 (抗体 H16L1-A);
或编码重链可变区基因的核苷酸序列如 SEQ ID No.49所示, 编码轻 链可变区基因的核苷酸序列如 SEQ ID No.50所示 (抗体 H16L1-B);
或编码重链可变区基因的核苷酸序列如 SEQ ID No.59所示, 编码轻 链可变区基因的核苷酸序列如 SEQ ID No.60所示 (抗体 H18L1-A)。
本发明还提供了含有所述编码基因的重组载体或表达系统。所述重组 载体的原始载体为 pACT2或 pET27b。
本发明还提供了所述的抗人乳头瘤病毒 L1蛋白抗体在制备预防、 治 疗人乳头瘤病毒相关疾病的药物中的应用。
本发明还提供了所述的抗人乳头瘤病毒 L1蛋白抗体在制备检测人乳 头瘤病毒相关疾病的试剂中的应用。
本发明抗人乳头瘤病毒 L1蛋白抗体能特异地结合人乳头瘤病毒 L1蛋 白, 检测 HPV感染的细胞。 其中, 利用全长重组的人源抗人乳头瘤病毒 L1蛋白抗体能检测 HPV感染的细胞, 因此, 可利用本发明的抗人乳头瘤 病毒 L1蛋白抗体来制备检测人乳头瘤病毒相关疾病的试剂。
另外, 研究发现, HPV抗体能够有效地中和 HPV病毒对人细胞的感 染性, 因此, 可利用本发明高亲和力的抗人乳头瘤病毒 L1蛋白抗体制备 药物, 以预防、 治疗人乳头瘤病毒相关疾病。
其中, 在上述两种应用中, 所述人乳头瘤病毒相关疾病可以为任何与 人乳头瘤病毒(HPV)感染有关(直接或间接引发)的疾病, 如子宫颈癌、 肛门癌、 阴道癌、 阴茎癌、 口腔癌、 咽喉癌等。 进一步的, 所述人乳头瘤 病毒相关疾病具体为人乳头瘤病毒引起的宫颈上皮内瘤变或宫颈癌,更进 一步的, 所述人乳头瘤病毒相关疾病可以为与 16型人乳头瘤病毒感染相 关的疾病(此时可应用抗体 H16L1-A或 H16L1-B)或 18型人乳头瘤病毒
感染相关的疾病 (此时可应用抗体 H18L1-A)。
与现有技术相比, 本发明的有益效果为:
本发明的抗人乳头瘤病毒 L1蛋白抗体是能用于检测 HPV感染,也可 以用于预防、 治疗人体疾病的全人源抗人乳头瘤病毒 L1蛋白抗体, 亲和 力高, 能特异结合人乳头瘤病毒 L1蛋白, 因此, 可制成药物或试剂, 有 效检测、 预防和治疗 HPV感染引起的疾病。 附图说明
图 1为本发明单链抗体的结构示意图。
图 2为本发明在昆虫 s©细胞所表达的人乳头瘤病毒 16型 L1蛋白在 SDS-PAGE后 Coomassie兰染色结果。 其中, 第 1道: 空载体感染后 s© 细胞总蛋白; 第 2道: 含 HPV16-L1的载体感染后 sf9细胞总蛋白; 第 3 道: 纯化后的 HPV16型 L1蛋白。
图 3为本发明抗人乳头瘤病毒 L1蛋白单链抗体 #H16L1-A的 ELISA 检测结果。
图 4为本发明抗人乳头瘤病毒 L1蛋白抗体 YFH16L1-A的 Western Blot结果; 其中, 第 1道: 正常宫颈细胞样本; 第 2道: CIN2期患者的 宫颈脱落细胞; 第 3道: CIN3期患者的宫颈脱落细胞; 第 4道: 宫颈癌 患者的宫颈脱落细胞。
图 5为本发明抗人乳头瘤病毒 L1蛋白单链抗体 #H16L1-B的 ELISA 检测结果。
图 6为本发明抗人乳头瘤病毒 L1蛋白单链抗体 #H18L1-A的 ELISA 检测结果。
图 7为本发明抗人乳头瘤病毒 L1 蛋白抗体 YFH16L1-B的 Western Blot结果; 其中, 第 1道: 正常宫颈细胞样本; 第 2道: CIN2期患者的 宫颈脱落细胞; 第 3道: CIN3期患者的宫颈脱落细胞; 第 4道: 宫颈癌 患者的宫颈脱落细胞。
图 8为本发明在昆虫 s©细胞所表达的人乳头瘤病毒 18型 L1蛋白在 SDS-PAGE后 Coomassie兰染色结果; 其中, 第 1道: 空载体感染后 s© 细胞总蛋白; 第 2道: 含 HPV18-L1的载体感染后 sf9细胞总蛋白; 第 3
道: 纯化后的 HPV18型 LI蛋白。
图 9为本发明抗人乳头瘤病毒 L1蛋白抗体 YFH18L1-A的 Western Blot结果; 其中, 第 1道: 正常宫颈细胞样本; 第 2道: CIN2期患者的 宫颈脱落细胞; 第 3道: 宫颈癌患者的宫颈脱落细胞。 具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细说明。 实施例 1 抗人乳头瘤病毒 L1蛋白单链抗体的筛选
采用公开号为 CN 1444651 A的中国专利文献公开的方法构建人单链 抗体文库, 并在该人单链抗体文库中筛选抗人乳头瘤病毒 L1蛋白单链抗 体, 具体实施过程如下:
1扩增得到人抗体重链和轻链可变区 DNA
以来自人骨髓、 人胎肝、 人脾和人外周血白细胞的 poly A+ RNA (购 自 Clontech) 为模板, 禾 U用 oligo (dT)禾口随机引物 (random primers), 使 用逆向转录酶试剂盒 (购自 Clontech), 根据 Clontech试剂盒所提供的方 法指南, 将 poly A+ RNA逆向转录成 cDNA。
以上述 cDNA为模板, 利用一系列识别人抗体重链可变区 (VH) 和 轻链可变区 (VL) 基因的引物, 进行 PCR扩增得到人抗体中所有的重链 可变区和轻链可变区的 DNA序列。 一系列识别人抗体重链和轻链可变区 基因的引物序列如下:
第一组为扩增人抗体重链可变区 (VH) 基因的 5'-端引物 (SEQ ID No.ll~17), 包括:
VHlb: 5, - CCATACGATGTTCCAGATTACCAGGTGCAGCTGCAG
GAGTC(C/G)G-3,;
VH2b: 5, -CCATACGATGTTCCAGATTACCAGGTACAGCTGCAGC
AGTCA-3';
VH3b: 5,-CCATACGATGTTCCAGATTACCAGGTGCAGCTACAGCA
GTGG G-3,;
VH4b: ;, -CCATACGATGTTCCAGATTACGAGGTGCAGCTG(G/T T GGAG(A/T)C(C/T)-3,;
VH5b:
(A/G)CAGTCTGG-3,;
VH6b:
GGAGTCTG-3' ;
VH7b: ;, -CCATACGATGTTCCAGATTACCAGGTGCAGCTGGTG
(C/G)A(A/G)TCTGG-3 ';
第二组为扩增人抗体重链可变区 (VH)基因的 3'-端引物 (SEQ ID No.18-23 ), 包括:
VHlf: 5,-GCCGCCTGATCCACCACCGCCTGAGGAGAC(A/G)GTGA
CCAGGGTG-3' ;
VH2f: ;, -GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACC
AGGGTT-3' ;
VH3f: i'-GCCGCCTGATCCACCACCGCCTGAAGAGACGGTGACC
ATTGT-3';
VH4f: i'-GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACC
GTGGTCC-3' ;
VH5f: ;, -GCCGCCTGATCCACCACCGCCGGTTGGGGCGGATGC
ACTCC-3' ;
VH6f: 5,-GCCGCCTGATCCACCACCGCC(C/G)GATGGGCCCTTGG
TGGA(A/G)GC-3,;
第三组为扩增人抗体 λ-轻链可变区 (νλ)基因的 5,-端引物 (SEQ ID No.24-32), 包括:
VLlb: 5
TGACGCAGCCGCC-
VL2b:
(A/T)CAGCCAC-3,;
VL3b:
(A/G)CAGC(C/T)ACC-3,;
VL4b: 5,-GGCAGCGGTGGTGGAGGCAGTCAGCCTGTGCTGACT
CA(A/G)(C/T)C-3,;
VL5b:
AC(C/T)CAGGAGCC-3,;
VL6b:
GACTCAGCC(A/C)CC-3,;
VL7b:
TCAGGA(C/G)CC-3,;
VL8b:
GA(C/T)TCAGCCT-3,;
VL9b: ;, -GGCAGCGGTGGTGGAGGCAGTAATTTTATGCTGACTCA
GCCCC-3' ;
第四组为扩增人抗体 λ-轻链可变区 (νλ)基因的 3'-端引物 (SEQ ID No.33~34), 包括:
CTTGGTCC-3' ;
VL2f: 5, -GGGGTTTTTCAGTATCTACGAGAGGACGGTCAGCTGG
GTGC-3' ;
第五组为扩增人抗体 k-轻链可变区 (Vk)基因的 5'-端引物 (SEQ ID No.35~38), 包括:
VKlb:
(A/G/T)TGACCCAGTCTCC-3 ';
VK2b:
GAC(A/G)CAGTCTCC-3,;
VK3b:
(C/G/T)CAG(A/T)CTCC-3,;
VK4b: ;, -GGCAGCGGTGGTGGAGGCAGTGAAACGACACTCACG
CAGTCTC-3';
第六组为扩增人抗体 k-轻链可变区 (Vk)基因的 3'-端引物 (SEQ ID No.39~42), 包括:
VKlf: 5'-GGGGTTTTTCAGTATCTACGATTTGATTTCCACCTTGG
TCC-
VK2f:
GGTCC-3' ;
VK3f: -GGGGTTTTTCAGTATCTACGATTTGATATCCACTTTGG
TCC-3' ;
VK4f: -GGGGTTTTTCAGTATCTACGATTTAATCTCCAGTCGTG
TCC-3,。
扩增人抗体中的重链可变区 (VH)时,利用第一组引物与第二组引物的 组合, 即有 42个 PCR反应; 扩增人抗体中的 λ-轻链可变区 (νλ)时, 利用 第三组引物与第四组引物的组合, 即有 18个 PCR反应; 扩增人抗体中的 k轻链可变区 (Vk)时, 利用第五组引物与第六组引物的组合, 即有 16个 PCR反应。
第一组引物中包含有酵母双杂交载体 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. )多克隆位点的上游同源序列(下 划线部分);第四组和第六组引物中包含有酵母双杂交载体 pACT2多克隆 位点的下游同源序列 (下划线部分); 而第二组、 第三组和第五组引物中 包含有连接肽序列 (下划线部分), 连接肽用于连接抗体的重链可变区和 轻链可变区。
扩增时, PCR反应体系与反应条件均相同, PCR反应体系为:
PCR缓冲液 (10x ) 5.0 μL
5,-端引物 (ΙΟ μΜ) 1.0 μ∑
3,-端引物 (ΙΟ μΜ) L0 μ∑ cDNA底物 5.0 μΐ dNTP ( lO mM) 1.0 μΐ
Taq聚合酶 (Takara) 1.0 单位 水 (ddH20 ) 36 μΐ
将上述各组分混合均匀后置于 PCR仪中进行反应。反应条件为: 94 °C 解链 1分钟, 50°C退火 1分钟, 72 °C延伸 2.5分钟, 循环 30次。
2连接载体多克隆位点的同源序列和连接肽序列
( 1 ) 以步骤 1中 PCR所得的人抗体重链可变区 DNA为模板, 分别 以引物 7和引物 8为上游引物和下游引物, 序列为:
上游引物(引物 7, SEQ ID No.43 , 下划线部分为载体 pACT2多克隆 位点的上游同源序列) :
ATACGATGTTCCAGATTAC-3,;
下游引物(引物 8, SEQ ID No.44, 下划线部分为连接肽反链序列): 5 '-ACTGCCTCCACCACCGCT
GCCTGATCCACCACCGCC ,
进行 PCR扩增, 反应条件和体系同步骤 1。 反应完成后获得含 5'-载 体 pACT2多克隆位点的上游同源序列和 3'-连接肽反链序列的人抗体重链 可变区 DNA序列。
(2) 以步骤 1中 PCR所得的人抗体轻链可变区 DNA为模板, 以引 物 9和引物 10分别为下游引物和上游引物, 序列如下:
上游引物(引物 10, SEQ ID No.45, 下划线部分为连接肽顺链序列): 5'-GGCGGTGGTGGATCAGGCGGCGGAGGATCTGGCGGAGGTGG
CAGCGGTGGTGGAGGCAGT-3,;
下游引物(引物 9, SEQ ID No.46, 下划线部分为载体 pACT2多克隆 位点的下游同源序列):
5'-GAGATGGTGCACGATGCACAGTTGAAGTGAACTTGCGGGGT
TTTTCAGTATCTACGA-3 ';
进行 PCR扩增, 反应条件和体系同步骤 1。 反应完成后获得含 3'-载 体 pACT2多克隆位点的下游同源序列和 5'-连接肽顺链序列的人抗体轻链 可变区 DNA序列。
3连接单链抗体
将步骤 2中 PCR扩增得到的含载体 pACT2多克隆位点的同源序列和 连接肽序列的人抗体重链可变区 DNA和轻链可变区 DNA混合, 以该混 合 DNA为模板,分别以引物 7和引物 9为上游引物和下游引物,进行 PCR
如图 1所示, 反应完成后得到包括人抗体重链可变区 DNA序列、 轻 链可变区 DNA序列、 载体 pACT2多克隆位点同源序列以及连接肽 DNA 序列的单链抗体 (scFv) DNA。
4构建人单链抗体基因文库
将步骤 3得到的单链抗体 (scFv) DNA与经限制性内切酶 (Mm HI 和 Eco RI) 处理后的酵母双杂交载体 pACT2按照原 Clontech公司提供的 方法(Yeast Protocol Handbook, PT3024-1 )共同转入酵母菌株 Υ187(ΜΑΓ% 画3-52, his3-200, ade2-101, lys2-801, trp 1-901, leu2-3, 112, gaU Δ, gal80A, met-, URA3:: GAL1 UAS-GAL1 TATA-lac Z,MEL1 )内, 经细胞内同源重组后将单 链抗体 DNA整合到 pACT2载体上, 从而得到酵母双杂交单链抗体文库, 单链抗体 DNA片段与 pACT2载体上的 Gal4激活区域(Activation Domain, AD ) 融合在一起。
为检查该单链抗体基因文库的质量, 从中随机挑出了 21个克隆, 对 插入片段进行测序分析。 分析结果表明, 所有克隆都包括与 Gal4融合在 一起的单链抗体 DNA片段, 而且所有单链抗体 DNA序列都是独特的。
经同源重组而得到的酵母双杂交单链抗体基因文库中抗体 DNA拷贝 数大约有 I X 108个, 可应用于酵母双杂交技术筛选特定的抗体。
5筛选抗体
( 1 ) 抗体筛选
1 ) 以人乳头瘤病毒 16型 (HPV16 ) L1蛋白作为抗原进行筛选 使用人乳头瘤病毒 16型(HPV16 ) L1蛋白 (衣壳主要蛋白:)作为抗原, 对酵母双杂交单链抗体基因文库进行抗体筛选。
将编码人乳头瘤病毒 HPV16型 L1蛋白的 DNA (序列见 GenBank的 ID号 U89348 )重组到载体 pGBKT7中,构建 pGBK-H16Ll。pGBK- H16L1 编码 Gal4 DNA结合区域 (Binding Domain, 简称 BD), 并在其 C端融合 了人乳头瘤病毒 HPV16型 L1蛋白。
在编码人乳头瘤病毒 HPV16型 L1蛋白的 DNA序列得到验证后, 将 PGBK-H16L1质粒 DNA转化入酵母菌株 AH109 (MATa, trp 1-901, leu2-3,
112, 画3-52, his3-200, gal4 Δ, gal80A, LYS2: : GAL1 GAL1皿 -HIS3, GAL2uAS- GAL2TATA-ADE2, URA3:: MEL1 -MELln-lac Z ); 带有 pGBK-H16Ll质粒的 AH109酵母可在不含色氨酸的合成培养基 (SD/-W) 上生长。
将等量的含 PGBK-H16L1的 MA 型酵母细胞 (AH109菌株) 与含 单链抗体基因文库的 ΜΑΓ 型酵母细胞 (Y187菌株) 进行共同培养, 使 此二型细胞交配结合。 由于载有单链抗体基因文库的 pACT2 载体含有 L 2基因, 且 pGBK-H16Ll包含 > 7基因, 因此, 包含两种质粒的酵母 细胞可以生长在不含亮氨酸和丝氨酸的酵母合成培养基 (SD/-LW)。
存在单链抗体 scFv与 L1蛋白相互作用的酵母细胞会激活整合在菌株 基因组中的报告基因 ADE2和 H 3 ,从而使得酵母细胞可以生长在缺乏腺 嘌呤, 组氨酸, 亮氨酸和色氨酸的培养基 (SD/-AHLW) 上, 并在该平板 培养基上形成菌落。
2 ) 以人乳头瘤病毒 18型 (HPV18 ) L1 蛋白作为抗原进行筛选使用 人乳头瘤病毒 18型 (HPV18 ) L1蛋白 (衣壳主要蛋白:)作为抗原, 对酵母 双杂交单链抗体基因文库进行抗体筛选。
将编码人乳头瘤病毒 HPV18型 L1蛋白的 DNA (序列见 GenBank的 ID号 X05015 )重组到载体 pGBKT7中,构建 pGBK-H18Ll。pGBK- H18L1 编码 Gal4 DNA结合区域 (Binding Domain, 简称 BD), 并在其 C端融合 了人乳头瘤病毒 HPV18型 L1蛋白。
在编码人乳头瘤病毒 HPV18型 L1蛋白的 DNA序列得到验证后, 将 PGBK-H18L1质粒 DNA转化入酵母菌株 AH109 (MATa, trpl-901, leu2-3, 112, 画3-52, his3-200, gal4 A, gal80A, LYS2: : GAL1^-GAL1TATA-H1S3, GAL2uA&- GAL2TATA-ADE2, URA3:: MEL1 -MELln-lac Z ); 带有 pGBK-H18Ll质粒的 AH109酵母可在不含色氨酸的合成培养基 (SD/-W) 上生长。
将等量的含 PGBK-H18L1的 MA 型酵母细胞 (AH109菌株) 与含 单链抗体基因文库的 ΜΑΓ 型酵母细胞 (Y187菌株) 进行共同培养, 使 此二型细胞交配结合。 由于载有单链抗体基因文库的 pACT2 载体含有 L 2基因, 且 pGBK-H18Ll包含 > 7基因, 因此, 包含两种质粒的酵母
细胞可以生长在不含亮氨酸和丝氨酸的酵母合成培养基 (SD/-LW)。
存在单链抗体 scFv与 L1蛋白相互作用的酵母细胞会激活整合在菌株 基因组中的报告基因 ADE2和 H 3 ,从而使得酵母细胞可以生长在缺乏腺 嘌呤, 组氨酸, 亮氨酸和色氨酸的培养基 (SD/-AHLW) 上, 并在该平板 培养基上形成菌落。
(2 ) 特异性抗体筛选
1 ) β半乳糖苷酶分析
由于存在 scFv与 L1蛋白相互作用的细胞也会激活整合在菌株基因组 中的另一报告基因 lac Z, 因此检测酵母细胞内 β半乳糖苷酶是否表达可判 断酵母细胞内 scFv/Ll蛋白的存在。
以人乳头瘤病毒 16型 (HPV16 ) L1蛋白作为抗原进行筛选时, 在筛 选培养基 (SD/-AHLW) 上总共挑选出 67个菌落, 以人乳头瘤病毒 18型 (HPV18 ) L1蛋白作为抗原进行筛选时, 在筛选培养基(SD/-AHLW)上 总共挑选出 52个菌落, 使用 β半乳糖苷酶检测法检测 lac Z表达。 检测方 法如下:
①将存在 scFv/Ll 蛋白相互作用的酵母接种到筛选培养基
( SD/-AHLW) 平板上生长;
②将酵母菌落转移到 Whatman五号滤纸上;
③将带有酵母细胞菌落的滤纸浸入液氮之中, 使细胞破裂;
④将滤纸从液氮中取出, 并置于 30°C的烘箱内; 重复步骤(3 )和(4 ) 两次;
⑤将滤纸铺于适量的 X-gal溶液中,并置于 37°C的温箱内约 15分钟; 若菌落处显示蓝色, 表明为 β半乳糖苷酶阳性, 即 toc Z基因被激活表达。
其中, X-gal溶液配方如下:
16.1 g/L Na2HP04«7H20;
5.50 g/L NaH2P04«H20;
0.75 g/L KC1;
0.246 g/L MgS04 ·7Η20;
35 mg/L X-gal (5-bromo-4-chloro-3-indolyl- -D-galactopyranoside); 5 ηιΜ β-巯基乙醇; pH 7.0。
检测结果: 上述以人乳头瘤病毒 16型(HPV16) L1蛋白作为抗原进行 筛选时, 挑选出的 67个菌落中, 有 37个是 β半乳糖苷酶阳性, 以人乳头瘤 病毒 18型 (HPV18) L1蛋白作为抗原进行筛选时, 挑选出的 52个菌落中, 有 23个是 β半乳糖苷酶阳性, 表明在这些菌落中报告基因 tocZ被激活了。
2) 特异结合分析
①以人乳头瘤病毒 16型 (HPV16) L1蛋白作为抗原筛选的阳性菌落 对上述 37个 β半乳糖苷酶阳性菌落的 scFv进行特异性分析, 以验证单 链抗体 scFv是否特异性地与人乳头瘤病毒 L1蛋白结合。
从上述 37个 β半乳糖苷酶阳性的酵母中提取含有 scFv的 pACT2质粒 DNA,再分别与 pGBKT7空载体 DNA、 pGBKT-IRa质粒 DNA、 pGBKT-Lam 质粒 DNA (编码 Gal4 DNA结合区域并在其 C端融合有人核纤层蛋白 C) 共 同转化到 AH109酵母细胞内; 转化后的酵母细胞先铺于 SD/-LW平板培养 基上生长, 然后转移到 SD/-AHLW平板培养基上; 对长出的菌落作 β半乳 糖苷酶分析, 验证为阳性的菌落视为含有非特异性的 scFv, 将其剔除。
经上述特异性分析后, 得到两个对人乳头瘤病毒 L1蛋白具有特异性 的单链抗体, 分别编号为 #H16L1-A和#^61^ , 使用 ABI 自动测序仪 对该单链抗体进行测序分析, 结果显示:
#H16L1-A的重链可变区的 DNA序列如 SEQ ID No.3所示。
RTVDSGYDFIPDWFHPWGQGTLVTVSS;
下划线部分依次为三个高变区 CDRH1、 CDRH2、 CDRH3 (SEQ ID No. 5-7);
#H16L1-A的轻链可变区的 DNA序列如 SEQ ID No.4所示。
YVFGTGTKLTVL;
下划线部分依次为三个高变区 CDRL1、 CDRL2、 CDRL3 (SEQ ID
Νο·8~10)。
#H16L1-B的重链可变区的 DNA序列如 SEQ ID No.49所示。
#H16L1-B的重链可变区的氨基酸序列 (SEQ ID No.47) 为:
OVTLKESGPALVKPTOTLTLTCTFSGFSLTTTGMCVNWIROSPGKPLE
YCARIHRREQGRHWDFDYWGQGTPVTVSS;
下划线部分依次为三个高变区 CDRH1、 CDRH2、 CDRH3 (SEQ ID No.51~53 );
#H16L1-B的轻链可变区的 DNA序列如 SEQ ID No. 50所示。
VFGEGTQLTV;
下划线部分依次为三个高变区 CDRL1、 CDRL2、 CDRL3 (SEQ ID Νο·54~56)。
②以人乳头瘤病毒 18型 (HPV18) L1蛋白作为抗原筛选的阳性菌落 对上述 23个 β半乳糖苷酶阳性菌落的 scFv进行特异性分析, 以验证单 链抗体 scFv是否特异性地与人乳头瘤病毒 HPV18型的 L1蛋白结合。
从上述 23个 β半乳糖苷酶阳性的酵母中提取含有 scFv的 pACT2质粒 DNA,再分别与 pGBKT7空载体 DNA、 pGBKT-IRa质粒 DNA、 pGBKT-Lam 质粒 DNA (编码 Gal4 DNA结合区域并在其 C端融合有人核纤层蛋白 C) 共 同转化到 AH109酵母细胞内; 转化后的酵母细胞先铺于 SD/-LW平板培养 基上生长, 然后转移到 SD/-AHLW平板培养基上; 对长出的菌落作 β半乳 糖苷酶分析, 验证为阳性的菌落视为含有非特异性的 scFv, 将其剔除。
经上述特异性分析后, 得到一个对人乳头瘤病毒 L1蛋白具有特异性 的单链抗体, 编号为 #H18L1-A, 使用 ABI 自动测序仪对该单链抗体进行 测序分析, 结果显示:
#H18L1-A的重链可变区的 DNA序列如 SEQ ID No.59所示。
#H18L1-A的重链可变区的氨基酸序列 (SEQ ID No.57) 为:
AVYYCTKIIAPDAFDIWGQGTMVTVSS;
下划线部分依次为三个高变区 CDRH1、 CDRH2、 CDRH3 ( SEQ ID Νο·61~63 ) ;
#H18L1-A的轻链可变区的 DNA序列如 SEQ ID No.60所示,
GPGTKVEIK;
下划线部分依次为三个高变区 CDRL1、 CDRL2、 CDRL3 ( SEQ ID Νο·64~66)。 实施例 2重组人乳头瘤病毒 HPV16型 L1蛋白、 HPV18型 L1蛋白表达 及兔子抗血清的制备
重组人乳头瘤病毒 HPV16型 L1蛋白的制备参照彭清林等 (彭清林, 张婷, 范东升, 郑伟, 谢喜秀, 许于飞, 许雪梅 (2009) "HPV 18 L1病 毒样颗粒的表达纯化及其豚鼠抗血清的制备。" 基础医学与临床, 29: 1039-1043)。 简述之: 将 HPV16型 L1编码 DNA片段根据 s©昆虫细 胞的密码子频率调整 HPV16型 L1编码 DNA的密码子进行优化, 优化后 的编码 DNA 插入 pFastBac Dual 载体 Xba I 位点中获得重组载体 /?FastBac-HPV16Ll o 通过转化大肠杆菌 DHlOBac 感受态细胞获得重组 Bacmido 用重组 Bacmid转染昆虫 s©细胞, 27 °C恒温培养箱中培养 72 小时后收集细胞培养上清, 获得表达 HPV16型 L1蛋白的重组杆状病毒。 利用空斑法测定病毒滴度后, 以 MOI=0.1感染细胞进行病毒富集。 随后, 用高滴度的病毒液感染 s©昆虫细胞, SDS-PAGE分析 (见图 2)。
重组病毒大量感染 sf9 细胞, 72 h后,收集感染细胞,超声破碎, 4 °C, 12000 rpm离心 10分钟,取上清液,经 CsCl密度梯度超速离心法纯化 VLP (Shi W, Liu J, Huang Y 等 (2001) Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses. J
Virol 75: 10139- 10148.)。最后用透析液( l O mM HEPES, 150 mM NaCl)透析 3小时得到纯化的 HPV16型 LI蛋白 (见图 2)。
兔子抗 HPV16型 L1血清的制备按照 Harlow等的传统方法 (Harlow E, 禾口 Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory 出版社, 第 53页至第 138页)。取纯化后的 HPV 16 L1 蛋白稀 释至 400 微升后与等体积弗氏佐剂充分混匀乳化, 分别于 0、 4、 8和 12 周皮下多点注射免疫兔子。 于最后一次免疫后 1周进行静脉取血, ELISA 检测血清抗体滴度, 滴度达到预期后进行取血, 收集血清。
HPV18型 L1蛋白表达及兔子抗血清的制备方法同 HPV16型 L1蛋白, 结果如图 8所示。 实施例 3特异性检测
1 单链抗体表达与纯化
将单链抗体 #H16L1 -A 的编码基因克隆到表达载体 pET27b(+)中, 构 建得到 pET27b-H16Lla;
将 pET27b-H16Lla 转化入表达细菌 E.coli BL21 (DE3), 并按照 Novagen公司提供的方法用 IPTG ( 0.5 mM ) 诱导表达; 表达出的目标蛋 白中, scFv的 N端是 pelB序列, pelB序列可将表达后的 scFv分泌到 BL21 (DE3)的周质腔 (periplasmic space ) 中; scFv的 C端含有一个 HSV 标记物和一个 6 X His标记物, 方便目标蛋白的纯化;
应用 Qiagen公司所提供的方法方便地用 Ni-NTA柱分离纯化得到抗人 乳头瘤病毒 HPV16型 L1蛋白的单链抗体。
单链抗体#11161^-:6、 #H18L1 -A 的表达与纯化方法可参照单链抗体 #H16L1-A的方法,将单链抗体 #H16L1-A的编码基因对应的替换为单链抗 体#11161^-:6或#11181^- 的编码基因即可。
2 ELISA测试单链抗体 #11161^- 和#11161^-:6对 HPV16型 L1蛋白的 特异性
昆虫细胞 sf9表达并纯化后的 HPV16型 L1蛋白见上述实施例 2。 ELISA测试方法如下:
( 1 ) 用 HPV16型 LI蛋白包被 96-孔板, 在 2-8°C过夜;
(2) 包被后的 96-孔板再用 SuperBlock作封闭处理;
(3 ) 将单链抗体 #H16L1-A在 0.02% BSA中作系列稀释后加入已包 被有 HPV16 L1蛋白的 96-孔中, 与 HPV16 L1蛋白结合;
(4)将 96-孔板清洗后,加入稀释 5000倍的鼠抗 HSV标记物的抗体, 用来检测结合了的单链抗体;
(5 ) 将 96-孔板清洗后, 加入稀释 10000倍的山羊抗鼠 IgG抗体-辣 根过氧化物酶偶联物;
(6) 将 96-孔板最终清洗后, 应用辣根过氧化物酶底物 TMB试剂作 显色处理;
(7) 用 0.5M的硫酸终止反应, 并检测 450 nm的吸收光谱。
单链抗体#11161^-8的特异性检测方法同单链抗体#11161^-入,仅将单 链抗体 #H16L1-A替换为单链抗体#11161^-:6即可。
检测结果如图 3、 图 5 所示, 表明单链抗体 #H16L1-A 和单链抗体 #H16L1-B均能有效地结合 HPV16型 L1蛋白。
3 ELISA测试单链抗体 #H18L1-A对 HPV18型 L1蛋白的特异性 昆虫细胞 sf9表达并纯化后的 HPV18型 L1蛋白见上述实施例 2。 ELISA测试方法同上述" 2 ELISA测试单链抗体 #H16L1-A P#H16Ll-B 对 HPV16型 L1蛋白的特异性"。
检测结果如图 6所示, 表明单链抗体 #H18L1-A能有效地结合 HPV18 型 L1蛋白。 实施例 4抗人乳头瘤病毒 L1蛋白抗体检测 HPV感染宫颈脱落细胞
委托北京义翘神州生物技术有限公司制备全长重组人源抗人乳头瘤 病毒 L1蛋白抗体 YFH16L1-A、YFH16L1-B和 YFH18L1-A。YFH16L1-A 、 YFH16L1-B和 YFH18L1-A均为 IgGl型, YFH16L1-A的可变区序列与单 链抗体 #H16L1-A的可变区序列相同, YFH16L1-B的可变区序列与单链抗 体#11161^-8 的可变区序列相同, YFH18L1-A 的可变区序列与单链抗体 #YFH18L1-A的可变区序列相同。
正常宫颈细胞样本、 宫颈上皮内瘤样病变 II期 (即 CIN2期)、 宫颈
上皮内瘤样病变 III期 (即 CIN3期)、 和宫颈癌患者的宫颈脱落细胞均来 自浙江大学医学院附属妇产科医院, 并获得患者知情同意。
Western Blot分析全长人源抗人乳头瘤病毒 L1蛋白抗体。 约 104宫颈 脱落细胞经 SDS-PAGE样品液处理后加样到 10%的 SDS-PAGE凝胶电泳 系统。 SDS-PAGE电泳后, 把蛋白转移到硝酸纤维素膜上。 然后, 该膜用 3%脱脂奶粉 -磷酸缓冲液 (pH 7.4)封闭 30分钟后,加入 1 ng/ml的全长重组 人源抗人乳头瘤病毒 L1 蛋白抗体 (YFH16L1-A、 YFH16L1-B 或 YFH18L1-A)室温摇 60分钟。 该膜用磷酸缓冲液 (pH 7.4)清洗后, 加入辣 根过氧化物酶标记山羊抗人 IgG ( Zymed公司)(1:4000稀释)。 Western Blot 发光检测试剂盒购自 PIERCE公司。
检测结果如图 4、 图 7、 图 9所示, 表明抗人乳头瘤病毒 L1蛋白抗体 YFH16L1-A, YFH16L1-B和 YFH18L1-A均可特异性的检测被 HPV感染 的宫颈脱落细胞。
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ccatacgatg ttccagatta ccaggtacag ctgcagcagt ca
<210> 13
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<212> DNA
<213> 人工序列
<400> 13
ccatacgatg ttccagatta ccaggtgcag ctacagcagt ggg
<210> 14
<211> 42
<212> DNA
<213> 人工序列
<400> 14
ccatacgatg ttccagatta cgaggtgcag ctgktggagw cy
<210> 15
<211> 44
<212> DNA
<213> 人工序列
<400> 15
ccatacgatg ttccagatta ccaggtccag ctkgtrcagt ctgg
<210> 16
<211> 43
<212> DNA
<213> 人工序列
<400> 16
ccatacgatg ttccagatta ccagrtcacc ttgaaggagt ctg <210> 17
<211> 44
<212> DNA
<213> 人工序列
<400> 17
ccatacgatg ttccagatta ccaggtgcag ctggtgsart ctgg
<210> 18
<211> 43
<212> DNA
<213> 人工序列
<400> 18
gccgcctgat ccaccaccgc ctgaggagac rgtgaccagg gtg
<210> 19
<211> 43
<212> DNA
<213> 人工序列
<400> 19
gccgcctgat ccaccaccgc ctgaggagac ggtgaccagg gtt
<210> 20
<211> 42
<212> DNA
<213> 人工序列
<400> 20
gccgcctgat ccaccaccgc ctgaagagac ggtgaccatt gt
<210> 21
<211> 44
<212> DNA
<213> 人工序列
<400> 21
gccgcctgat ccaccaccgc ctgaggagac ggtgaccgtg gtcc <210> 22
<211> 41
<212> DNA
<213> 人工序列
<400> 22
gccgcctgat ccaccaccgc cggttggggc ggatgcactc c
<210> 23
<211> 42
<212> DNA
<213> 人工序列
<400> 23
gccgcctgat ccaccaccgc csgatgggcc cttggtggar gc
<210> 24
<211> 44
<212> DNA
<213> 人工序列
<400> 24
ggcagcggtg gtggaggcag tcagtctgts btgacgcagc cgcc
<210> 25
<211> 43
<212> DNA
<213> 人工序列
<400> 25
ggcagcggtg gtggaggcag ttcctatgwg ctgacwcagc cac
<210> 26
<211> 44
<212> DNA
<213> 人工序列
<400> 26
ggcagcggtg gtggaggcag ttcctatgag ctgayrcagc yacc <210> 27
<211> 41
<212> DNA
<213> 人工序列
<400> 27
ggcagcggtg gtggaggcag tcagcctgtg ctgactcary c
<210> 28
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<212> DNA
<213> 人工序列
<400> 28
ggcagcggtg gtggaggcag tcagdctgtg gtgacycagg agcc
<210> 29
<211> 44
<212> DNA
<213> 人工序列
<400> 29
ggcagcggtg gtggaggcag tcagccwgkg ctgactcagc cmcc
<210> 30
<211> 44
<212> DNA
<213> 人工序列
<400> 30
ggcagcggtg gtggaggcag ttcctctgag ctgastcagg ascc
<210> 31
<211> 42
<212> DNA
<213> 人工序列
<400> 31
ggcagcggtg gtggaggcag tcagtctgyy ctgaytcagc ct <210> 32
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<212> DNA
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<400> 32
ggcagcggtg gtggaggcag taattttatg ctgactcagc ccc
<210> 33
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<212> DNA
<213> 人工序列
<400> 33
ggggtttttc agtatctacg ataggacggt sascttggtc c
<210> 34
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<212> DNA
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ggggtttttc agtatctacg agaggacggt cagctgggtg c
<210> 35
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<212> DNA
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<400> 35
ggcagcggtg gtggaggcag tgacatccrg dtgacccagt ctcc
<210> 36
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<212> DNA
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ggcagcggtg gtggaggcag tgaaattgtr wtgacrcagt ctcc <210> 37
<211> 44
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<400> 37
ggcagcggtg gtggaggcag tgatattgtg mtgacbcagw ctcc
<210> 38
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ggcagcggtg gtggaggcag tgaaacgaca ctcacgcagt etc
<210> 39
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<212> DNA
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<400> 39
ggggtttttc agtatctacg atttgatttc caccttggtc c
<210> 40
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ggggtttttc agtatctacg atttgatctc cascttggtc c
<210> 41
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gacatccggc tgacccagtc tccttccacc ctgtctgcct ctgtcggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt ggctggttgg cctggtatca acagagacca 120 gggagagccc ctaagctcct gatctatggg gcatctactt tggaaagtgg ggtcccatca 180 aggttcagcg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttttta ctgccaacag tataataatt atccgtggac gttcggccca 300
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Claims
1、一种抗人乳头瘤病毒 L1蛋白抗体,包括重链可变区和轻链可变区, 其特征在于,
所述重链可变区的三个高变区 CDRH1、 CDRH2、 CDRH3 的氨基酸 序列分别为: GGSIRSGDY、 SYSGT、 TVDSGYDFIPDWFHP, 所述轻链 可变区的三个高变区 CDRL1、 CDRL2、 CDRL3 的氨基酸序列分别为 SGSNSNIGNSYVH, RN QRPS, AAWADSLGTYV;
或所述重链可变区的三个高变区 CDRH1、 CDRH2、 CDRH3 的氨基 酸序列分别为: GFSLTTTGM、 DWDDD、 IHRREOGRHWDFDY, 所述轻 链可变区的三个高变区 CDRL1、 CDRL2、 CDRL3 的氨基酸序列分别为 SGSSSNLGSNFVY, SNVQRPS, AAWDDSLDVLV;
或所述重链可变区的三个高变区 CDRH1、 CDRH2、 CDRH3 的氨基 酸序列分别为: GDSVSSNSNTA, YYRSKWY, IIAPDAFDI, 所述轻链可 变区的三个高变区 CDRL1、 CDRL2、 CDRL3 的氨基酸序列分别为 RASOSISGWLA, GASTLES, QQYNNYPWTo
2、 如权利要求 1所述的抗人乳头瘤病毒 L1蛋白抗体, 其特征在于, 所述重链可变区的氨基酸序列如 SEQ ID No.l所示, 所述轻链可变区的氨 基酸序列如 SEQ ID No.2所示;
或所述重链可变区的氨基酸序列如 SEQ ID No.47所示, 所述轻链可 变区的氨基酸序列如 SEQ ID No.48所示;
或所述重链可变区的氨基酸序列如 SEQ ID No.57所示, 所述轻链可 变区的氨基酸序列如 SEQ ID No.58所示。
3、 如权利要求 1所述的抗人乳头瘤病毒 L1蛋白抗体, 其特征在于, 所述抗人乳头瘤病毒 L1蛋白抗体为全抗体或全抗体的抗原结合部分。
4、 如权利要求 3所述的抗人乳头瘤病毒 L1蛋白抗体, 其特征在于, 所述的全抗体为 IgGl型。
5、 如权利要求 3所述的抗人乳头瘤病毒 L1蛋白抗体, 其特征在于, 所述的抗原结合部分为 Fab片段、 Fab'片段、 F(ab':>2片段或单链抗体。
6、编码如权利要求 2所述的抗人乳头瘤病毒 L1蛋白抗体的基因, 其 特征在于, 编码重链可变区基因的核苷酸序列如 SEQ ID No.3所示, 编码
轻链可变区基因的核苷酸序列如 SEQ ID No.4所示;
或编码重链可变区基因的核苷酸序列如 SEQ ID No.49所示, 编码轻 链可变区基因的核苷酸序列如 SEQ ID No.50所示;
或编码重链可变区基因的核苷酸序列如 SEQ ID No.59所示, 编码轻 链可变区基因的核苷酸序列如 SEQ ID No.60所示。
7、 含有如权利要求 6所述的基因的重组载体。
8、 含有如权利要求 6所述的基因的表达系统。
9、如权利要求 1~5任一所述的抗人乳头瘤病毒 L1蛋白抗体在制备预 防、 治疗人乳头瘤病毒相关疾病的药物中的应用。
10、 如权利要求 1~5任一所述的抗人乳头瘤病毒 L1蛋白抗体在制备 检测人乳头瘤病毒相关疾病的试剂中的应用。
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CN201310720040.9A CN103694345B (zh) | 2013-12-23 | 2013-12-23 | 抗人乳头瘤病毒l1蛋白抗体及其编码基因和应用 |
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