丙肝病毒NS3-4A基因的重组杆状病毒穿梭载体的构建

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。

Construction and identification of recombinant baculovirus transfer vector for expressing NS3-4A protein of HCV

Aim To amplify HCV NS3-4A gene and construct the recombinant baculovirus vector containing NS3-4A of HCV.Methods HCV RNA was extracted from HCV patients’ serum and NS3-4A gene was amplified by RT-PCR with the designed primer.Then the NS3-4A fragment was digested with EcoR I and HindIII and ligased with the pFastbacDual vector of baculovirus.After transformation,the recombinant plasmid was identified by PCR and double endnuclease digestion.Results A 2.2Kb fragment was obtained from amplification,the sequence was confirmed to be HCV NS3-4A gene by sequence analysis.PCR and double endonucleases results confirmed that the HCV NS3-4A gene recombinant expression vector pFB-NS3-4A was successfully constructed.Conclusion Recombinant baculovirus transfer vector pFB-NS3-4A was successfully constructed,which can be applied to construct the HCV vaccine based on baculovirus.