丙型肝炎病毒全长cDNA模板的构建及鉴定

Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient

目的 构建具有功能的丙型肝炎病毒 (HCV)全长cDNA克隆。方法 应用长模板RT PCR法扩增 1份上海地区HCV感染者血清的RNA(基因型为 1b) ,分段扩增、融合拼接成 9 2kb的基因片段 ,克隆入含有HCV基因两端非编码区序列的载体作为模板。为检测此过程是否发生对特定变异株的选择 ,分析 4个独立克隆的HVR1序列。对原核细胞中表达的HCV核心蛋白、NS3蛋白酶及解旋酶 ,以蛋白印迹实验确证其免疫反应性。并构建NS3 4A SEAP表达系统检验NS3的蛋白酶活性。结果 获得丙型肝炎病毒全长cDNA模板。不同克隆间HVR1序列存在较大差异 ,提示长模板RT PCR所制备HCVcDNA具有HCV基因准种 (quasispecies)的特性。该模板编码基因在原核细胞内得到高效表达 ,并具有良好的免疫反应性。在NS3 4A SEAP表达系统内 ,NS3可切割、释放其下游的SEAP ,具有蛋白酶活性。结论 本工作为构建全长功能性HCVcDNA模板及感染性克隆奠定了基础。

Objective To construct the full-length complementary DNA of HCV genome from an HCV infected patient. Methods Four HCV gene fragments (1.6,3.5,2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment,which was further cloned into a cassette vector with fixed 5′ and 3′ termini of HCV to make the full-length cDNA. The cDNA′s heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions. The prokaryotic expressed Core,NS3 protease,NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis. And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system. Results The cDNA covered the near full-length of HCV genome from the patient's serum. The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA. The prokaryotic expressed viral proteins could be identified by patient serum. In the NS3/4A-SEAP system,NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media. Conclusion These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.