【作者】 魏薇；

【导师】 何金生；

【作者基本信息】 安徽医科大学 ， 免疫学， 2009， 硕士

【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。更多还原

【Abstract】 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.更多还原