| | | 不同缺失突变的丙型肝炎病毒核心蛋白基因在大肠杆菌中的表达
| | 中药炮制论文 作者：沈才飞，白雪帆，牟丹蕾，黄长形
【关键词】 丙型肝炎病毒；核心蛋白；突变；原核表达
Expressions of different deletion mutated HCV core protein genes in E.coli
【Abstract】 AIM: To construct the recombinant plasmids expressing fulllength HCV core protein gene and 3 different deletion mutated hepatitis core protein genes and to express them in E.coli. METHODS: Entire HCV core protein gene and 3 different deletion mutated gene fragments from HCV core protein which respectively coded 1-191aa, 1-59aa plus 81-191aa, 1-169aa, 1-59aa plus 81-169aa were amplified by PCR and cloned into expression vector pRSETA, thus forming different recombinant plasmids （pRSETAC573, pRSETAC510, pRSETAC507, pRSETAC444）, which were transformed into E.coli BL21 （DE3） pLysS and induced by IPTG. SDSPAGE and Western blot were used to test and identify expressed 6×Hisfusion proteins. RESULTS: Restriction analysis and sequencing confirmed that the 4 recombinant plasmids expressing entire HCV core protein gene and 3 different deletion mutated hepatitis core protein genes were successfully constructed. Both SDSPAGE and Western blot analysis showed entire HCV core protein gene and 3 different deletion mutated hepatitis core protein genes were expressed, and the respective molecular weights of the products were about 21×103, 19×103, 19×103 and 16.5×103. The fusion protein production accounted approximately for 20% of total bacterial protein. CONCLUSION: Different deletion mutated HCV core protein genes were expressed efficiently in E.coli.
【Keywords】 hepatitis C virus; core proteins; mutation; prokaryotic expression
【摘要】 目的中药炮制论文： 构建丙型肝炎病毒（HCV）全长及3种不同缺失突变的中药炮制论文核心蛋白基因的原核表达载体，并在大肠杆菌中表达. 方法：用PCR方法扩增基因型为1b型的HCV核心蛋白的全长及3种不同缺失突变的基因片段，对应氨基酸分别为C573：1-191aa，C510:1-59aa+81-191aa，C507:1-169aa，C444:1-59aa+81-169aa. 将其克隆到高效表达载体pRSETA中构成重组质粒（pRSETAC573，pRSETAC510，pRSETAC507，pRSETAC444），转化大肠杆菌BL21（DE3）pLysS. IPTG诱导表达6×His融合蛋白，表达产物经SDSPAGE及Western blot检测和鉴定. 结果： 经酶切鉴定及测序证实全长及3种不同缺失突变的核心蛋白基因的原核表达载体构建正确. 经SDSPAGE及Western blot显示在Mr约为21×103, 19×103, 19×103, 16.5×103处出现融合表达条带. 融合蛋白的表达量约占菌体蛋白总量的20％. 结论：全长及3种不同缺失突变的丙型肝炎病毒核心蛋白基因均在大肠杆菌中得到有效表达.
【关键词】 丙型肝炎病毒；核心蛋白；突变；原核表达
【中图号】 R512.630.27
0引言
丙型肝炎病毒（hepatitis C virus, HCV）感染已成为全球性的社会公共卫生问题，迄今尚无有效的预防性疫苗. HCV核蛋白基因相对保守［1］并且能诱导CTL反应及ADCC，有可能用于开发HCV预防及治疗性疫苗. 我室曾扩增羧基末端的20个氨基酸和（或）第60~80位氨基酸多肽基因缺失的核蛋白基因，并在真核细胞中表达成功，初步证实突变掉上诉两个肽断能提高机体免疫反应［2-3］. 为进一步探讨不同缺失突变核心蛋白对免疫反应的影响，我们构建了HCV核心基因不同缺失突变基因片段的原核表达载体并在大肠杆菌中进行了表达.
1材料和方法
1.1材料
1.1.1质粒与菌株源质粒pCI573, pCI510, pCI507, pCI444系本室将不同缺失突变的核心蛋白基因克隆入真核表达载体pCIneo中构建而成，其中分别插入了自西安地区患者血清中克隆的1b型HCV完整的核心蛋白基因（573 bp）, 60～80位氨基酸缺失的核心蛋白基因片段（510 bp）, 羧基末端20位氨基酸缺失的核心蛋白基因片段（507 bp）, 同时突变掉60～80位氨基酸和羧基末端20位氨基酸的核心蛋白基因片段（444 bp）. 原核表达载体pRSETA为Invitrogin公司产品，中间克隆载体pGEMT easy为Promega公司产品. E. coli JM109由本室保存,BL21为Invitrogin公司产品.
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