人类8型疱疹病毒小衣壳蛋白ORF65基因的原核表达及其生物信息学分析  

The prokaryotic expression for small capsid ORF65 gene of HHV-8 and its bioinformatics analysis

作　　者：热依汗.谢以提 克迪亚[2] 阿斯哈尔[3] 王星[4] 何方平[5] 温浩[4] 何斌[6] 

机构地区：[1]新疆医科大学第一附属医院临检中心,新疆乌鲁木齐830000 [2]新疆医科大学第三附属医院检验科,新疆乌鲁木齐830000 [3]新疆医科大学黄河路中医院戒毒中心,新疆医科大学新疆乌鲁木齐830000 [4]新疆医科大学第一附属医院中心实验室,新疆乌鲁木齐830000 [5]新疆医科大学感染性疾病科,新疆乌鲁木齐830000 [6]美国芝加哥大学伊利诺医学院免疫与微生物中心实验室,美国芝加哥il606127344

出　　处：《新疆医科大学学报》2007年第2期100-103,共4页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区自然科学基金资助项目(200421122)

摘　　要：目的:构建人类8型疱疹病毒(HHV-8)小衣壳蛋白(ORF65)基因重组原核表达质粒并诱导表达,获得其重组融合蛋白。方法:软件设计引物,在引物两端添加特异性酶切位点,以pGEM-Teasy/ORF65为模板,PCR扩增基因序列,克隆入原核表达载体pET-41a中,构建原核表达质粒pET41a-ORF65,转化大肠杆菌(E.coli)DH5x,经酶切、测序鉴定其插入序列的正确性,转入E.coli BL21(DE3),并诱导表达,采用聚丙烯酰胺凝胶电泳(SDS-PAGE)方法以及序列分析软件DNAMAN 5.0、Vector NTI Suite 6、Primer Primier 5对表达蛋白进行生物信息学分析。结果:测序结果表明所构建pET41a-ORF65原核表达质粒连接正确,SDS-PAGE表明ORF65基因获得成功表达,生物信息学分析显示该蛋白含有1个N糖基化位点,2个蛋白激酶C磷酸化位点,4个胆碱激酶Ⅱ磷酸化位点,4个N肉豆蔻酰化位点,1个酰胺化位点。BlastP数据库的相似性检索发现有与之有较高同源性的蛋白。结论:成功表达HHV-8病毒小衣壳蛋白ORF65,该蛋白与疱疹病毒衣壳蛋白的同源性较高,是疱疹病毒衣壳蛋白家族中的一员。Objective： To express the small capsid ORF65 Gene of human herpesvirus 8（HHV-8） in E. coli, and analysis its bioinformatics. Methods： ORF65 was amplified by PCR, using pGEM-Teasy/ORF65 as template and subsequently inserted into prokaryotic expression vector pET-41a. And then identified by double enzyme digesting and sequencing. The recombinant plasmids pET41a-ORF65 was transformed into competent E. coli BL21 （DE3） cells. The fusion protein was induced. The fusion protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PEGE）. Its bioinformatics was analyzed by DNAMAN 5.0, Vector NTI Suite 6, Primer Primier 5. Results： Double enzyme digesting and sequencing showed the insert target DNA was 534 bp, a 43 kD fusion protein was exhibited on SDS-PAGE when the recombinant bacteria was induced by IPTG. The bioinformatics analysis demonstrated that the protein had an amidation site, two phosphorylation sites of protein kinase C, four phosphorylation sites of choline kinase Ⅱ , four N muscade acylation sites and an amidation site. Conclusion： The small capsid ORF65 gene of HHV-8 was correctly expressed in E. coli BL21 cells. The bioinformatics analysis demonstrate that homology of the protein and herpesvirus capsid protein is high, the protein is one of the herpesvirus capsid protein family.

关 键 词：人类疱疹病毒8型 小衣壳蛋白ORF65基因 原核表达 生物信息学分析 

分 类 号：R752[医药卫生—皮肤病学与性病学]