丙型肝炎病毒NS3蛋白的原核表达及多克隆抗体制备

Prokaryotic expression of Hepatitis C Virus (HCV) NS3 protein and preparation of polyclonal antibody

摘要:

提高对丙型肝炎患者实验室检测的灵敏度和特异性，对相关人群进行筛查和早期诊断，是控制丙型肝炎病毒 (HCV) 流行与传播的有效措施。为了建立更为可靠的HCV诊断方法，通过采用PCR方法从J6/JFH1 2a型病毒中克隆出HCV ns3基因片段，将其连接到pET-28a载体上，重组载体pET-28a-ns3转化大肠杆菌BL21 (DE3) 后诱导表达，以10% SDS-PAGE进行鉴定，获得表达的NS3重组蛋白分子量为72 kDa。将纯化的NS3蛋白免疫BALB/c小鼠，第4次免疫后采集血液并分离血清进行抗体活性鉴定，小鼠抗体效价为1∶256 000。进一步的Western blotting和间接免疫荧光结果显示，以重组NS3蛋白免疫小鼠制备的多克隆抗体可以很好地识别HCV感染Huh7.5.1细胞中的NS3蛋白，为下一步开展单克隆抗体制备和检测试剂盒研制工作奠定了基础。

Abstract:

To increase detection sensitivity and specificity on hepatitis C virus (HCV) is vital for prevention and controlling of the disease. To establish a more reliable detection method for HCV diagnosis, the full gene fragment of ns3 (non-structural protein of HCV) from recombinant plasmid of J6/JFH1 2a was amplified and then connected into the pET-28a prokaryotic expression vector, and the latter was subsequently transformed into Escherichia coli BL21 (DE3) to have the target protein expression. As a result, a protein with a molecular weight of 72 kDa was obtained and visualized in 10% SDS-PAGE. The purified NS3 protein was used as immunogen to inoculate BALB/c mice and the sera was collected after the fourth immunization. The antibody titer of serum is determined to be about 1:256 000 with ELISA. Western blotting and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native NS3 protein in Huh 7.5.1 cells infected with HCV. These findings may provide basis for further preparation of monoclonal antibodies against NS3 and the development of related detection kit.