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

发布时间：2017-12-31 08:38

本文关键词：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　出处：《安徽医科大学》2009年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective: human Respiratory Syncytial virus (RSVV) is widely distributed all over the world. It is the most important virus pathogen leading to severe lower respiratory tract infection in infants. The mechanism of immune protection after virus infection is not clear, and there is no specific prevention and cure method. Among the 11 proteins encoded by RSV. Fusion glycoprotein F) and adhesion glycoprotein attachment (glycoprotein). G) is the only two neutralizing antigens, and F protein has high antigenic homology among different subtypes of RSV. This study attempts to construct a large capacity natural human phage antibody library using phage display technology. Single chain Fv fragment (scFv) of RSV F protein was screened. To lay a foundation for the prevention and specific treatment of RSV infection. Methods: peripheral blood lymphocytes from 10 healthy donors were isolated and total RNAs were extracted from them. The VHV L gene of human antibody was amplified by RT-PCR. The scFv gene was cloned into pCANTAB5E phagocyte vector and transformed into E. coli TG1 receptive cells. A large phage antibody library was obtained, and then the recombinant bacteriophage was saved by auxiliary phage M13K07. The phage scFv, which specifically recognizes RSV F protein, was enriched in the antibody library, and the positive clones were screened by phage ELISA method. The positive clones were infected with E. coli HB2151 and the soluble scFv of RSV F protein was prepared by IPTG induction. The method of Western and Dot blot was used to analyze and identify. Results A complete set of human antibody VH (about 350bp) was amplified by using the method of: RT- PCR. The scFv gene (about 750bp) was successfully spliced into pCANTAB5E vector by SOE-PCR in vitro. A large human natural phage phage scFv library with a capacity of 1.8 脳 10 ~ 7 was constructed. After five rounds of biological washing, the specific RSV F recognition scFv was 108-fold enriched. Eighteen positive clones were screened by phage ELISA. The clone E4 with the highest OD value was sequenced and analyzed by Kabat database. Western and Dot blot analysis showed that its gene was highly homologous to the variable region gene of human immunoglobulin, and the single chain antibody was successfully expressed. Conclusion: a large capacity natural human phage scFv library was constructed, from which scFv, which could specifically recognize RSV F, was successfully screened, and the soluble expression and preliminary identification were carried out. It lays a foundation for the further study of biological activity.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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