抗猪瘟病毒单链抗体的原核表达及活性鉴定

Prokaryotic expression and activity identification of anti-Classical swine fever virus single chain antibody

为了制备抗猪瘟病毒(CSFV)单链抗体(scFv)并鉴定其生物学活性,试验以编码特异性抗CSFV scFv基因序列为基础,按照大肠杆菌密码子偏爱性优化并合成scFv基因,将其克隆入pCzn1表达载体,转化大肠杆菌BL21(DE3)PlysS感受态细胞,经IPTG诱导表达后进行Ni-IDA亲和层析纯化、SDS-PAGE检测和Western-blot鉴定,通过间接免疫荧光(IFA)和间接ELISA验证蛋白抗原的活性。结果表明:密码子优化合成的抗CSFV scFv基因全长768 bp,在大肠杆菌中主要以包涵体形式表达,其分子质量约28.0 ku,Western-blot检测显示纯化后的目的蛋白能被抗His标签单克隆抗体特异性识别,IFA和ELISA检测证实纯化复性后的蛋白可与CSFV发生特异性结合反应,且存在明显的浓度依赖性。说明应用原核表达系统可高效制备抗CSFV scFv。

Abstarct:To prepare the single-chain antibody(scFv)against viral Classical swine fever virus(CSFV)and identify its biological function,in this study,based on encoding specific anti-CSFV scFv gene sequence,the experiment optimized and synthesized scFv gene according to the codon preference of escherichia coli,cloned it into pCzn1 expression vector,and transformed into BL21(DE3)PlysS and induced by IPTG for the expression of the recombinant protein.The expressed product was purified with Ni-IDA and identified by SDS-PAGE and Western-blot assay,and its antigen binding activity was tested by IFA and ELISA.The results showed that a 768 bp anti-CSFV scFv coding gene sequence was optimized and synthesized,the recombinant protein was in the form of inclusion bodies with a molecular weight of about 28.0 ku expressed in E.coli.Western-blot analysis showed that the purified target protein could be specifically identified by anti-His-tagged monoclonal antibody,and IFA and ELISA confirmed that the purified protein had a specific binding reaction with CSFV.It indicated that the prokaryotic expression system could efficiently prepare CSFV scFv.