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目的通过克隆丙肝病毒非结构蛋白NS3-4A基因,构建表达HCV-NS3-4A基因的重组杆状病毒穿梭载体。方法设计合成HCV NS3-4A基因全长的特异性引物,应用逆转录-多聚酶链反应(RT-PCR)扩增非结构蛋白区NS3-4A基因片段,经限制性内切酶EcoR I和Hind III双酶切后克隆到pFastbacDual上,并转化到大肠杆菌DH5α中,最后经PCR和双酶切鉴定转化菌落。结果扩增得到的基因片段长度为2.2kb,该片段成功克隆在杆状病毒穿梭载体pFastBacDual上,经测序证实为HCV NS3-4A基因,这表明HCV-NS3A基因重组杆状病毒穿梭载体构建成功。结论成功构建了表达HCV NS3-4A基因的杆状病毒穿梭载体,这将为下一步HCV杆状病毒载体疫苗的制备打下基础。
Objective To construct a recombinant baculovirus shuttle vector expressing HCV-NS3-4A gene by cloning the nonstructural protein NS3-4A of hepatitis C virus. Methods Specific primers for the synthesis of HCV NS3-4A gene were designed and used to amplify the NS3-4A gene fragment in the non-structural protein region by reverse transcription-polymerase chain reaction (RT-PCR). The restriction endonucleases EcoR I and Hind III Double digested cloned into pFastbacDual and transformed into E. coli DH5α, and finally identified by PCR and double-digested transformed colonies. Results The amplified fragment was 2.2kb. The fragment was successfully cloned into the baculovirus shuttle vector pFastBacDual and verified by sequencing to be HCV NS3-4A gene, indicating that the recombinant baculovirus shuttle vector of HCV-NS3A gene was successfully constructed. Conclusion The successful construction of baculovirus shuttle vector expressing HCV NS3-4A gene will lay the foundation for the further preparation of HCV baculovirus vector vaccine.