乙型肝炎核心抗原基因的合成及其原核表达载体的构建  

Gene synthesis and construction of prokaryotic expression vector of hepatitis B core antigen

作　　者：王丽[1] 刘金波[2] 段春燕[1] 龚舒[1] 何涛[1] 

机构地区：[1]泸州医学院基础医学院医学分子生物学实验室,泸州646000 [2]郑州大学第一附属医院肛肠外科,郑州450052

出　　处：《郑州大学学报（医学版）》2009年第6期1242-1245,共4页Journal of Zhengzhou University(Medical Sciences)

摘　　要：目的:合成乙型肝炎病毒核心抗原(HBcAg)基因并构建原核表达载体。方法:根据大肠杆菌偏嗜性密码子,利用SyntheticGeneDesigner软件对HBcAg基因进行密码子优化,分成24条相互重叠的寡核苷酸片段,采用PCR法扩增获得完整的HBcAg基因,经BamHⅠ和XhoⅠ双酶切后定向克隆到pET30a(+),得pET30a(+)-HB-cAg,经PCR扩增、酶切及测序鉴定后,定点突变法改正错误位点。之后将重组子转入BL21(DE3),IPTG诱导表达,SDS-PAGE电泳分析表达情况,WesternBlot检测蛋白抗原性。结果:成功构建了重组质粒pET30a(+)-HBcAg,SDS-PAGE分析显示目的蛋白主要以包涵体的形式在大肠杆菌表达,表达效率64%左右;WesternBlot显示表达产物具有较好的抗原性。结论:成功合成HBcAg基因并构建了原核表达载体。Aim:To synthesize gene and construct prokaryotic expression vector of hepatitis B core antigen(HBcAg).Methods:By selecting the biased codon in E.coli,the HBcAg gene was optimized by software of Synthetic Gene Designer and was divided into 24 oligonucleotide fragments.The complete target fragment was synthesized using assembly PCR and cloned into pET30a(+)vector at NdeⅠ/XhoⅠ site.After it was analyzed with restriction endonuclease digestion and DNA sequencing analysis,the gene which contained error was corre...

关 键 词：乙型肝炎病毒核心抗原 基因合成 定点突变 原核表达 

分 类 号：Q78[生物学—分子生物学]