丙型肝炎病毒非结构3区基因片段的克隆表达及抗原纯化鉴定

Cloning and Expression of NS_3 Gene Fragment of HCV and Identification of the Purified Products

通过逆转录ＰＣＲ技术，从中国江苏省丙型肝炎病人血清中扩增克隆了丙型肝炎病毒（ＨＣＶ）的非结构３区的部分基因片段（Ｃ３３）。ＤＮＡ序列分析证实，该片段全长８４２ｂｐ。在合成的一对逆转录ＰＣＲ引物上，上游引物增加了ＮｃｏⅠ酶切位点（内含起始密码子ＡＴＧ），下游引物上增加了ＳａｌⅠ酶切位点及终止密码子ＴＡＡ，将克隆的Ｃ３３基因片段克隆至表达载体ｐＢＶ２２１内，获得了表达Ｃ３３抗原的工程菌，表达Ｃ３３抗原分子过为３０ｋＤ，经尿素裂解纯化、分子筛分离纯化及复性后进行离子交换和反相亲和层析纯化，获得的重组蛋白经ＥＬＢＡ和免疫印迹等证实有较好的抗原性和特异性。

he NS3 gene fragment of HCV was cloned from the serum of hepatitis C patients in Jiangshuprovince by RT-PCR.DNA sequencing shows the cloned NS3 cDNA contains 842bp,NcoI site and sal-I site were added respectively to the upetream and downstream primers.The cDNA was cut with NcoIand SalI,and then cloned into the NcoI and SalI sites in plasmid pBV221.The recombinant plasmidwas transformed into DH5a for gaining the E,coli expressing NS3 protein,Expressed NS3 protein(30kD)was about l7% of total proteins of the E.coli,and the purified NS3 protein by Sephacryl S-200 and IgG-Sepharose 4B chromatography(or DEAE Sepharose Fast Flow)has good antigenicity andspecificity.