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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/JFH12a型病毒中克隆出HCV ns3基因片段,将其连接到p ET-28a载体上,重组载体p ET-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 k Da。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。
To improve the sensitivity and specificity of laboratory tests on patients with hepatitis C, screening and early diagnosis of related populations is an effective measure to control the spread and spread of hepatitis C virus (HCV). In order to establish a more reliable HCV diagnostic method, the HCV ns3 gene fragment was cloned from J6 / JFH12a virus by PCR and ligated into the pET-28a vector. The recombinant vector p ET-28a-ns3 was transformed into Escherichia coli After induced with BL21 (DE3), the recombinant protein was identified by 10% SDS-PAGE. The molecular weight of NS3 recombinant protein was 72 kDa. BALB / c mice were immunized with the purified NS3 protein. After the 4th immunization, blood was collected and serum was separated for antibody activity identification. The titer of the mouse antibody was 1:256 000. Further Western blotting and indirect immunofluorescence showed that the polyclonal antibodies raised in mice immunized with the recombinant NS3 protein could well recognize the NS3 protein in Huh7.5.1 cells infected by HCV. In order to further develop the monoclonal antibody preparation and detection reagent Box research and development laid the foundation.