抗鸡传染性支气管炎病毒核蛋白单链抗体的构建及初步鉴定

Preparation of a single-chain variable fragment against the IBV nucleocapsid protein

为了构建鸡传染性支气管炎病毒(IBV)核蛋白(N)单链抗体(scFv),本研究通过提取杂交瘤细胞株6H3的总mRNA,采用RT-PCR法扩增出抗IBVN蛋白抗体的重链和轻链可变区基因,利用重叠延伸PCR将其重链和轻链通过一条寡核苷酸链连接起来扩增了单链抗体基因,并将其克隆于原核表达载体pET-30a,构建重组质粒,转化E.coliBL21(DE3),经IPTG诱导表达。对表达产物进行纯化复性后,用夹心ELISA和westernblot检测表明scFv可与传染性支气管炎病毒核蛋白发生特异性结合。

A single chain variable fragment （scFv） was amplified from the hybridoma cell line 6H3 specific to IBV nucleoeap- sid protein by using splice-overlap extension polymerase chain reaction （SOE-PCR） through the flexible （Gly（4）Ser） （3） linker. The scFv gene was clone into the prokaryotic expression vector pET-30a, transformed into E. coli BL21 （DE3）, and protein ex- pression was induced by IPTG. The recombinant protein was purified using ProBondTM Purification System and was refolded by urea dialysis. Sandwich enzyme-linked immunosorbent assay （ELISA） and western blotting were performed to detect the reactivity between the scFv and IBV. The results indicated that scFv could react specifically with IBV nucleocapsid protein.