呼吸道合胞病毒融合蛋白人源性单链抗体的构建及初步鉴定
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摘要
目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染最重要的病毒病原。病毒感染机体后的免疫保护机制尚未明确,也无特异性防治方法。在RSV所编码的11种蛋白中,融合糖蛋白(fusion glycoprotein,F)和粘附糖蛋白(attachment glycoprotein,G)是仅有的两种中和抗原,而F蛋白在RSV不同亚型间具有高度的抗原同源性,是主要的交叉保护性抗原。本研究尝试利用噬菌体展示技术构建大容量天然人源性噬菌体抗体库,从中筛选出RSV F蛋白的人源性单链抗体(single chain Fv fragment, scFv),为RSV感染的预防及特异性治疗等研究奠定基础。
方法:分离10名健康供者外周血淋巴细胞,从中提取细胞总RNA,通过RT-PCR扩增人抗体VH、VL基因,并利用SOE-PCR将其拼接组装成scFv基因。将scFv基因克隆入pCANTAB5E噬菌粒载体,通过电转化E.coli TG1感受态细胞,获得大容量噬菌体抗体库,再经辅助噬菌体M13K07挽救重组噬菌粒。利用生物淘洗方法,使抗体库中特异性识别RSV F蛋白的噬菌体scFv得到富集,并利用噬菌体ELISA方法筛选出阳性克隆,阳性克隆进行核酸序列分析。将阳性克隆噬菌体感染E.coli HB2151,经IPTG诱导,制备RSV F蛋白的可溶性单链抗体,并以Western及Dot blot方法进行初步分析鉴定。
结果:RT- PCR法扩增出全套人抗体VH(约350 bp)、VL(约320 bp)片段,利用SOE-PCR方法在体外成功拼接成scFv基因(约750 bp)并克隆入pCANTAB5E噬菌粒载体,构建了库容为1.8×107的大容量人源性天然噬菌体scFv库。经过五轮生物淘洗,特异性识别RSV F的scFv得到了108倍的富集,噬菌体ELISA筛选出18株阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western及Dot blot分析表明单链抗体已成功表达。
结论:构建了大容量天然人源噬菌体scFv库,从中成功筛选出能特异性识别RSV F的scFv,并进行了可溶性表达和初步鉴定,为进一步的生物学活性研究奠定了基础。
Objective:The worldwide spread human respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness in infants and children. The mechanism of host immune protection against RSV infection remains unknown. So far no safe and effective vaccines are available. Among the encoded 11 viral proteins by RSV genome, the fusion glycoprotein (F) and attachment glycoprotein (G) are the only two neutralization antigens. F protein owns highly conserved amino-acid identity between different group’s viruses, which is the most important target antigen in vaccine project, elicits cross-protective immunity. In this research, the human single-chain Fv fragment (scFv) antibody library was constructed by phage display technology and the specific scFvs against RSV F protein were obtained by screening the human scFv library.
Methods: Peripheral blood lymphocytes (PBL) were separated from ten healthy donors, and the total RNA was extracted from the PBL. The variable heavy (VH) and variable light (VL) genes were amplified by RT-PCR and then the scFv genes, obtained through SOE-PCR, were cloned into the vector pCANTAB5E, and electroporated into competent E.coli TG1 cells to prepare a scFv phage display library. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for RSV F were enriched after five rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. The gene of the positive clone was identified by nucleic acid sequence analysis. After infecting E.coli HB2151 with the positive phage clone, soluble scFv was prepared, and then confirmed by Western and Dot blots.
Result: The VH (370 bp) and VL (320 bp) genes were amplified by RT-PCR and then the scFv (750 bp) genes were obtained through SOE-PCR. After the scFv genes were cloned into the vector pCANTAB5E and electroporated into competent E.coli TG1 cells, a scFv phage display library containing 1.8×107clones was gained. Recombinant phages specific for RSV F were enriched to 108 times after five rounds of biopanning and 18 antigen-positive clones were selected from the enriched clones by phage ELISA. DNA sequence analysis of the clone E4 with the highest OD indicated that the variable region gene of the clone was highly homologous with human immunoglobulin gene family. The expression of the scFv E4 was confirmed by Western and Dot blots.
Conclusion: From the human phage display library, the specific scFv for RSV F protein was selected and confirmed. This study laid a firm foundation for further investigation on the biological activity of the scFv.
方法:分离10名健康供者外周血淋巴细胞,从中提取细胞总RNA,通过RT-PCR扩增人抗体VH、VL基因,并利用SOE-PCR将其拼接组装成scFv基因。将scFv基因克隆入pCANTAB5E噬菌粒载体,通过电转化E.coli TG1感受态细胞,获得大容量噬菌体抗体库,再经辅助噬菌体M13K07挽救重组噬菌粒。利用生物淘洗方法,使抗体库中特异性识别RSV F蛋白的噬菌体scFv得到富集,并利用噬菌体ELISA方法筛选出阳性克隆,阳性克隆进行核酸序列分析。将阳性克隆噬菌体感染E.coli HB2151,经IPTG诱导,制备RSV F蛋白的可溶性单链抗体,并以Western及Dot blot方法进行初步分析鉴定。
结果:RT- PCR法扩增出全套人抗体VH(约350 bp)、VL(约320 bp)片段,利用SOE-PCR方法在体外成功拼接成scFv基因(约750 bp)并克隆入pCANTAB5E噬菌粒载体,构建了库容为1.8×107的大容量人源性天然噬菌体scFv库。经过五轮生物淘洗,特异性识别RSV F的scFv得到了108倍的富集,噬菌体ELISA筛选出18株阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western及Dot blot分析表明单链抗体已成功表达。
结论:构建了大容量天然人源噬菌体scFv库,从中成功筛选出能特异性识别RSV F的scFv,并进行了可溶性表达和初步鉴定,为进一步的生物学活性研究奠定了基础。
Objective:The worldwide spread human respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness in infants and children. The mechanism of host immune protection against RSV infection remains unknown. So far no safe and effective vaccines are available. Among the encoded 11 viral proteins by RSV genome, the fusion glycoprotein (F) and attachment glycoprotein (G) are the only two neutralization antigens. F protein owns highly conserved amino-acid identity between different group’s viruses, which is the most important target antigen in vaccine project, elicits cross-protective immunity. In this research, the human single-chain Fv fragment (scFv) antibody library was constructed by phage display technology and the specific scFvs against RSV F protein were obtained by screening the human scFv library.
Methods: Peripheral blood lymphocytes (PBL) were separated from ten healthy donors, and the total RNA was extracted from the PBL. The variable heavy (VH) and variable light (VL) genes were amplified by RT-PCR and then the scFv genes, obtained through SOE-PCR, were cloned into the vector pCANTAB5E, and electroporated into competent E.coli TG1 cells to prepare a scFv phage display library. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for RSV F were enriched after five rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. The gene of the positive clone was identified by nucleic acid sequence analysis. After infecting E.coli HB2151 with the positive phage clone, soluble scFv was prepared, and then confirmed by Western and Dot blots.
Result: The VH (370 bp) and VL (320 bp) genes were amplified by RT-PCR and then the scFv (750 bp) genes were obtained through SOE-PCR. After the scFv genes were cloned into the vector pCANTAB5E and electroporated into competent E.coli TG1 cells, a scFv phage display library containing 1.8×107clones was gained. Recombinant phages specific for RSV F were enriched to 108 times after five rounds of biopanning and 18 antigen-positive clones were selected from the enriched clones by phage ELISA. DNA sequence analysis of the clone E4 with the highest OD indicated that the variable region gene of the clone was highly homologous with human immunoglobulin gene family. The expression of the scFv E4 was confirmed by Western and Dot blots.
Conclusion: From the human phage display library, the specific scFv for RSV F protein was selected and confirmed. This study laid a firm foundation for further investigation on the biological activity of the scFv.
引文
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22. Dezorzova′-Tomanova′K, Molinkova′D, Pekarova M,et al. Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library[J]. Res Vet Sci, 2007, 83: 85–90
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2. Glezen WP, Taber LH, Frank AL, et al. Risk of primary infection and reinfection with respiratory syncytial virus[J]. Am J Dis Child , 1986, 140(6): 543–5466
3. Henrickson KJ, Hoover S, Kehl KS, et al. National disease burden of respiratory viruses detected in children by polymerase chain reaction[J]. Pediatr Infect Dis J , 2004, 23(1 ): 11–18
4. Simoes EA, Carbonell-Estrany X. Impact of severe disease caused by respiratory syncytial virus in children living in developed countries[J]. Pediatr Infect Dis J , 2003, 22(2 ): 13–18.
5.邓洁,钱渊,周汝南等.2000年冬-2006年春北京地区急性呼吸道感染患儿中呼吸道合胞病毒的监测[J].中华儿科杂志, 2006, 44(12): 924-927
6. Crowe, J.E., Jr., Current approaches to the development of vaccines against disease caused by respiratory syncytial virus (RSV) and parainfluenza virus (PIV). A meeting report of the WHO Programme for Vaccine Development. Vaccine, 1995, 13(4): 415-421.
7.吕治淋(编译),翟羽(审稿).儿童疫苗的现状与展望.国外医学流行病与传染病分册, 2000, 27(1): 36-39.
8. Ghildyal R, H.A., Jans DA. Central role of the respiratory syncytial virus matrix protein in infection[J]. FEMS Microbiol Rev, 2006,30(5): 692-705
9. Hoogenboom HR, Griffiths AD, Johnson KS, et al. Multi-subunit proteins on the surface of filamentous phage:Methodologies for displaying antibody (Fab) heavy and light chains[J]. Nucleic Acids Res, 1991, 19: 4133-4137
10. DeNardo SJ, DeNardo GL, DeNardo DG, et al. Antibody phage libraries for the next generation of tumor target ingradioimmunotherapeutics[J]. Clin Cancer Res,1999,5( 10): 3213-3218
11.Clackson T, Hoogenboom HR, Griffiths AD, et al. Making antibody fragmentsusing phage display libraries[J]. Nature, 1991, 352(6336): 624-628
12. Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface[J]. Science, 1985, 228(4705): 1315-l317
13. Orlandi R, Gussow DH, Jones PT. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction[J]. Proc Natl Acad Sci, USA, 1989, 86(10): 3833–3837
14. Marks JD, Hoogenboom HR, Bonnert TP. By-passing immunization: Human antibodies from V-gene libraries displayed on phage[J]. J Mol Biol, 1991, 222(3): 581-597
15. Winter G, Griffiths AD, Hawkins RE. Making antibodies by phage display technology[J]. Annu Rev Immunol, 1994, 12: 433-455
16. Knappik A, Ge L, Honegger A,et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides[J]. J Mol Biol, 2000, 296(1): 57-86
17. Hall, C.B. Respiratory syncytial virus and parainfluenza virus[J]. N Engl J Med, 2001, 344(25): 1917-1928
18. Walsh, E.E. and J. Hruska. Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein[J]. J Virol, 1983, 47(1): 171-177
19. Gold DV, Cardillo T, Goldenberg DM, et al. Localization of pancreatic cancer with radiolabeled monoclonal antibody PAM4[J]. Cri Rev Oncol Hematol, 2001, 39 (1-2): 147-154
20. Chatenaud L, Baudrihaye M, Chkoff N, et al. Restriction of the human in vivo response against mouse monoclonal antibody OKT3[J]. J Immunol, 1986, 137(3): 830-838
21. Adams GP, Schier R, McCall AM, et al. High affinity restricts the localization and tumor penetration of single-chain Fv antibody molecules[J]. Cancer Res, 2001, 6 1(12): 4750- 4755
22. Dezorzova′-Tomanova′K, Molinkova′D, Pekarova M,et al. Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library[J]. Res Vet Sci, 2007, 83: 85–90
23. Aizhi Zhao,Weijun Qin,Yueheng Han,et al. Isolation and identification of an scFv antibody against nucleocapsid protein of SARS-CoV[J]. Microbes Infect , 2007, 9: 1026-1033
24. Abi-Ghanem D, Suryakant D, David J,et al. Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites[J]. Vet Immunol Immunop, 2008, 121: 58–67
25. Huston JS, Levinson D, Mudget-Hunter M, et al. Protein engineering of antibody Binding sites:recovery of specificactivity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli[J]. Porc Natl Acad Sci USA ,1988, 85 (16): 5879-5883
26. Whitlow M, Bell BA, Feng SL, et al. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability[J]. Protein Eng, 1993 6( 8): 989-995
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