摘要
目的克隆乙型肝炎病毒X蛋白反式调节基因11(XTP11)剪切体,构建其原核表达载体,表达并纯化该蛋白。方法应用逆转录聚合酶链反应及生物信息学技术从HepG2细胞中提取cDNA为模板并扩增,意外获得XTP11基因的剪切体基因,选用pGEM-T载体进行T-A克隆,通过限制性酶切分析及测序鉴定,再将其亚克隆到原核表达载体pET-32a(+)中,转化BL21(DE3)宿主菌,经异丙基-B-D-硫代半乳糖苷(IPTG)诱导XTP11剪切体融合蛋白的表达,表达产物进行SDS-PAGE分析,考马斯亮蓝染色,以鼠His probe单克隆抗体进行Western blot分析鉴定证实表达蛋白的特异性,并纯化蛋白。结果成功扩增出XTP11剪切体基因,构建了pET-32a(+)XTP11剪切体原核表达载体,经IPTG诱导获得了大小约为69kD的重组蛋白。结论发现的HBV XTP11剪切体及其融合蛋白,为进一步研究其生物学功能奠定了基础。
Objective To construct prokaryotic expression vector carrying XTP11 spliced variant gene and to express and purify it. Methods Reverse transeription-polymerase chain reaction and bioinformatics technique were used to amplify XTP11 spliced variant from HepG2 cDNA template. The novel DNA fragment was ligated into pGEM-T cloning vector by TA cloning. After restriction enzyme digestion analysis and sequencing,the correct DNA fragment was inserted into inducible prokaryotic expression vector. The competent BL21 （DE3） E. coli was transformed,and then cultured and induced with isopropy-β-D-thiogalactoside （IPTG）. The expressed XTP11 spliced variant was confirmed with SDS-PAGE and Western blot hybridization,then the expressed product was purified. Results The spliced DNA fragment of XTP11 spliced variant was successfully amplified,and its expression vector was constructed. After induced with IPTG,the recombinant target protein of about 69kD was obtained. Conclusion A novel gene-binding protein spliced variant has been recognized,and its recombinant prokaryotic expression vector has been constructed. The expression and purification of recombinant XTP11 spliced variant gene will be valuable for the study on the biological function of the new gene.
出处
《实用肝脏病杂志》
CAS
2009年第4期248-251,273,共5页
Journal of Practical Hepatology
基金
北京市科委重大科技攻关项目(D08050700650803)
国家重点基础研究项目(973项目)2004(B518908)
关键词
乙型肝炎病毒
X蛋白反式调节基因
剪切体
基因克隆
原核表达
hepatitis B virus
X protein transregulate gene
Spliced variant
Gene cloning
Prokaryotic expression
