HCV核心抗原N-端片段生物素化表达质粒的构建及表达

目的构建HCV核心(C)抗原N-端1～130aa片段的生物素化表达质粒,并在大肠杆菌中进行表达。方法PCR扩增HCV C抗原N-端1～130aa片段的编码基因,拼接上生物素-蛋白连接酶底物肽序列(BSP),构建重组原核表达质粒pGEX4T-1-CN130BSP,转化大肠杆菌Rosetta,IPTG诱导表达。表达产物经SDS-PAGE后,电转至硝酸纤维素膜上,以抗HCVC抗原的单克隆抗体和辣根过氧化物酶标记的亲和素对表达产物进行分析。结果PCR扩增出约466bp的目的基因片段;质粒pGEX4T-1-CN130BSP经PCR及双酶切鉴定,与预期结果一致,测序鉴定基因无突变;SDS-PAGE显示表达产物相对分子质量约为42000,22℃诱导16h可溶性表达产物含量较高;表达的重组蛋白可被抗HCV C抗原的单克隆抗体所识别,并能特异性结合辣根过氧化物酶标记的亲和素。结论已成功构建了HCV C抗原N-端1～130aa片段生物素化表达质粒,并表达出带生物素标签的GST融合蛋白,为进一步研制HCV双抗原夹心检测试剂奠定了基础。

Construction and Expression of Biotinylated Expression Vector for Amino Acid Fragment at N-terminus of HCV Core Antigen

Objective To construct a biotinylated expression vector for 1 ～ 130 aa fragment at N-terminus of HCV core antigen.Methods The gene encoding 1 ～ 130 aa fragment at N-terminus of HCV core antigen was amplified by PCR and spliced to BirA substrate peptide(BSP)gene sequence.The constructed recombinant prokaryotic expression vector pGEX4T-1-CN130BSP was transformed to E.coli Rosetta for expression under induction of IPTG.The expressed product was identified by SDS-PAGE,then transferred to a nitrocellulose filter and analyzed with McAb against HCV core antigen and HRP-labeled avidin.Results A target gene fragment at length of 466 bp was amplified by PCR.Both PCR and restriction analysis proved that recombinant plasmid pGEX4T-1-CN130BSP was constructed correctly,and sequencing result showed no gene mutation.SDS-PAGE showed expressed protein with a relative molecular mass of about 42 000.After induction at 22℃ for 16 h,the expression level of soluble protein reached a peak value.The expressed product was recognized with McAb against HCV core antigen,and bound to HRP-labeled avidin specifically.Conclusion A biotinylated expression vector for 1 ～ 130 aa fragment at Nterminus of HCV core antigen was successfully constructed,and GST fusion protein with biotin labeling was expressed,which laid a foundation of further preparation of double antigen sandwich detection kit for HCV.