在大肠杆菌中以非融合蛋白形式高效表达丙型肝炎核心蛋白

High-level expression of hepatitis C virus core protein in E. coli in a non-fused form

应用聚合酶链反应（ＰＣＲ）技术，扩增出完整的丙型肝炎病毒核心蛋白基因，将其克隆于表达载体ｐＢＶ２２０ＰＲＰＬ启动子的下游，构建了重组质粒ｐＢＶ－ＨＣＣ；转化大肠杆菌后，以非融合会自方式表达了丙型肝炎病毒核心蛋白。ＳＤＳ－ＰＡＧＥ电泳显示，此蛋白的分子量约为２０ｋＤａ，表达量约占菌体蛋白的１１％；Ｗｅｓｔｅｒｎ印迹实验证明，此蛋白能与慢性丙型肝炎患者血清发生特异反应。序列分析结果表明，克隆于ｐＢＶ２２０的外源基因确为丙型肝炎核心蛋白基因。以非融合蛋白形式表达的丙型肝炎核心蛋白，对深入研究丙型肝炎核心蛋白的结构、功能及生物学活性将起积极作用。

By analyzing the published H C V RN A sequences with computer,the authors designed and synthesizedtwo HCV primers,which corresponded to the upstream and downstream of HCV core gene,respectively.The se-quenees of the two primers are: primer 1,5-GCGAATTCATGAGCACGAATCCTA-3(upstream);primer 2,5-CCGAATTCCCTGTTGCCTAGTTCA-3(downstream).Both primers contaied EcoR Ⅰsites,while to primer 2 is also introduced a termination codon TAG by base mutation.The predicted DNA fragment(516 bp) was amplified by polymerase chain reaction(PCR)frompGEM-HCV, which contains the HCV structure gene and part of the HCV non-structure gene. The PCR productwas purified and digested with EcoR Ⅰ and inserted into a high-level expression vector pBV220.Two positiverecombinants(designated pBV-HCV) were obtained by colony hybridization,restriction digestion andSDS-pAGE. Results from SDS-PAGE show that a specific protein with a molecular weight of 20 kDa at a levelof 11% of the total bacterial proteins appears in bacteria harboring pBV-HCV, while this protein is absent in con-trol bacteria harboring pBV220. Western blot analysis showed that this protein could be specifically recognized bythe serum from a patient with chronic hepatitis C virus infection,Sequence analysis of the amplified PCR productsalso confirms that we have successfully cloned and expressed the HCV core gene.