丙型肝炎病毒河北分离株基因组NS3 cDNA片段在大肠杆菌中的克隆与表达
Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli.
作者信息
Zhu Fen-Lu, Lu Hao-Ying, Li Zhuo, Qi Zhong-Tian
出版信息
World J Gastroenterol. 1998 Apr;4(2):165-168. doi: 10.3748/wjg.v4.i2.165.
AIM
To obtain greater antigenicity of HCV NS3 protein.METHODS:The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients.The DNA sequence was determined by dideoxy-mediated chain termination method using T7 polymerase.HCV NS3 protein was expressed in E. coli.RESULTS:Sequence analysis indicated that the HCV isolate of this study belongs to HCV-II; SDS-PAGE demonstrated an M(r) 23800 and an M(r) 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately.Western blotting and ELISA showed NS3 protein possessed greater antigenicity.CONCLUSION:Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.
目的
获得更高抗原性的丙型肝炎病毒（HCV）NS3蛋白。方法：通过逆转录聚合酶链反应从HCV感染患者血清中扩增HCV NS3 cDNA片段。采用双脱氧介导的链终止法，使用T7聚合酶测定DNA序列。HCV NS3蛋白在大肠杆菌中表达。结果：序列分析表明本研究中的HCV分离株属于HCV-II型；十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示有一条相对分子质量（M(r)）为23800和一条M(r)为22000的重组蛋白条带，分别占细菌总蛋白的14%和11%。蛋白质印迹法和酶联免疫吸附测定（ELISA）表明NS3蛋白具有更高的抗原性。结论：成功表达了重组HCV NS3蛋白，为开发HCV诊断试剂奠定了基础。
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