抗副溶血弧菌极鞭毛蛋白FlaB单链抗体的制备

发布时间：2018-05-01 23:53

  本文选题：极鞭毛蛋白 + 单链抗体　； 参考：《福建农林大学》2010年硕士论文

【摘要】： 副溶血弧菌是一种广泛存在于水环境中的革兰氏阴性嗜盐菌,更是一种常见的食源性病原菌,可污染多种水产品,并引起人的食物中毒。其鞭毛基因系统复杂,参与粘附作用,包括周身鞭毛和极性鞭毛两种形式。鞭毛基因系统包括约57个基因和3个开放阅读框,极性鞭毛系统与周身鞭毛系统的结构与装配成分并不分享。 本实验试图制备出一株抗其极鞭毛蛋白FlaB的高亲和力的单链抗体,希望建立一种更快速检测VP的检测手段。本实验首先从VP基因组中PCR扩增出flaB基因,并构建出重组质粒pET32a(+)-flaB。将该质粒转化宿主菌E. coli BL21( DE3 ),利用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达目的蛋白FlaB,经Ni2+-NTA纯化。 以纯化的目的蛋白作抗原免疫BalB/C小鼠, ELISA间接法检测抗血清的效价,效价达到后提取总RNA,通过RT-PCR反转录成cDNA、然后以cDNA模板PCR获得抗体重链可变区基因VH和轻链可变区基因VL,再用15氨基酸连接短肽Linker连接VH、VL组装成有活性的scFv基因,再将scFv基因构建到噬菌体载体pCANTAB-5E上,转化大肠杆菌TG1,实现与cpⅢ蛋白的融合表达,建立丝状噬菌体单链抗体展示库。 高库容量的噬菌体抗体库建立后,通过3~5轮的富集淘选和ELISA检测,最终选出一株亲和力高、稳定性强的单链抗体噬菌体,再将此噬菌体侵染大肠杆菌HB2151,实现单链抗体的可溶性表达。
[Abstract]:Vibrio parahaemolyticus is a gram-negative halophilic bacteria widely found in water environment and a common foodborne pathogen which can pollute many aquatic products and cause human food poisoning. The gene system of flagellum is complex and involved in adhesion, including peri-flagellum and polar flagellum. The flagellum gene system consists of about 57 genes and 3 open reading frames. The structure and assembly components of polar flagellum system and periflagellar system are not shared. In this study, a high affinity single chain antibody (scFv) against its flagellin FlaB was prepared in order to establish a more rapid method for the detection of VP. In this experiment, the flaB gene was amplified from the PCR of VP genome, and the recombinant plasmid pET32a (Flab) was constructed. The plasmid was transformed into E. coli BL21 (DE3), and the target protein FlaB was induced by isopropyl thiothio- 尾 -Dgalactoside. The target protein was purified by Ni2 NTA. The purified protein was used as antigen to immunize BalB/C mice. The titer of antiserum was detected by ELISA indirect method. After titer was reached, total RNAs were extracted, then transformed into cDNAs by RT-PCR reverse transcription, then VH and light chain variable region genes were obtained by cDNA template PCR. The active scFv gene was assembled with 15-amino acid ligated short peptide Linker. Then the scFv gene was constructed into the phage phage vector pCANTAB-5E and transformed into E. coli TG1. The fusion expression with cp 鈪，

本文编号：1831530