本文選題：極鞭毛蛋白 + 單鏈抗體��； 參考：《福建農(nóng)林大學(xué)》2010年碩士論文

【摘要】： 副溶血弧菌是一種廣泛存在于水環(huán)境中的革蘭氏陰性嗜鹽菌,更是一種常見的食源性病原菌,可污染多種水產(chǎn)品,并引起人的食物中毒。其鞭毛基因系統(tǒng)復(fù)雜,參與粘附作用,包括周身鞭毛和極性鞭毛兩種形式。鞭毛基因系統(tǒng)包括約57個(gè)基因和3個(gè)開放閱讀框,極性鞭毛系統(tǒng)與周身鞭毛系統(tǒng)的結(jié)構(gòu)與裝配成分并不分享。 本實(shí)驗(yàn)試圖制備出一株抗其極鞭毛蛋白FlaB的高親和力的單鏈抗體,希望建立一種更快速檢測(cè)VP的檢測(cè)手段。本實(shí)驗(yàn)首先從VP基因組中PCR擴(kuò)增出flaB基因,并構(gòu)建出重組質(zhì)粒pET32a(+)-flaB。將該質(zhì)粒轉(zhuǎn)化宿主菌E. coli BL21( DE3 ),利用異丙基硫代-β-D-半乳糖苷(IPTG)誘導(dǎo)表達(dá)目的蛋白FlaB,經(jīng)Ni2+-NTA純化。 以純化的目的蛋白作抗原免疫BalB/C小鼠, ELISA間接法檢測(cè)抗血清的效價(jià),效價(jià)達(dá)到后提取總RNA,通過RT-PCR反轉(zhuǎn)錄成cDNA、然后以cDNA模板PCR獲得抗體重鏈可變區(qū)基因VH和輕鏈可變區(qū)基因VL,再用15氨基酸連接短肽Linker連接VH、VL組裝成有活性的scFv基因,再將scFv基因構(gòu)建到噬菌體載體pCANTAB-5E上,轉(zhuǎn)化大腸桿菌TG1,實(shí)現(xiàn)與cpⅢ蛋白的融合表達(dá),建立絲狀噬菌體單鏈抗體展示庫(kù)。 高庫(kù)容量的噬菌體抗體庫(kù)建立后,通過3~5輪的富集淘選和ELISA檢測(cè),最終選出一株親和力高、穩(wěn)定性強(qiáng)的單鏈抗體噬菌體,再將此噬菌體侵染大腸桿菌HB2151,實(shí)現(xiàn)單鏈抗體的可溶性表達(dá)。
[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，

本文編號(hào)：1831530