摘要
目的:克隆乙型肝炎病毒e抗原结合蛋白未知功能新基因HBeBP4的剪切体基因HBeBP4A,构建其原核表达载体,诱导在大肠埃希菌中表达。方法:应用逆转录聚合酶链反应(RT-PCR)技术及生物信息学(bioinformat-ics)技术从HepG2细胞提取的cDNA模板中扩增获得HBeBP4基因的剪切体基因HBeBP4A,选用pGEM-T-easy载体进行TA克隆,通过PCR、限制性酶切分析及测序进行鉴定,再将其亚克隆到原核表达载体pET-32a(+)中,转化进BL21(DE3)宿主菌,IPTG诱导HBeBP4A融合蛋白的表达,表达产物进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,考马斯亮蓝染色,以抗-His的单克隆抗体进行Western blotting免疫印迹分析鉴定证实表达蛋白的特异性。结果:成功扩增出HBeBP4剪切体基因HBeBP4A,并将其插入pET-32a(+)原核表达载体,经BL21(DE3)受体菌转化、IPTG诱导、SDS-PAGE分析获得了HBeBP4A重组蛋白的表达,Western blotting证实了重组蛋白表达的特异性。结论:发现了乙型肝炎病毒e抗原结合蛋白4(HBeBP4)基因的剪切体HBeBP4A,构建了pET-32a(+)-HBeBP4A原核表达载体,并利用大肠埃希菌原核表达系统成功获得了pET-32a(+)-HBeBP4A重组蛋白的表达。
Objective： To clone a new gene, binding protein 4 spliced variant HBeBP4A of hepatitis B virus e antigen, and construct its recombinant prokaryotie expression vector, and induce the expression of recombinant protein in Escheriehia coll. Methods： Reverse transeription-polymerase chain reaction （RT-PCR） and bioinformatie technique were used to amplify HBeBP4A from HepG2 eDNA template. The novel DNA fragment was ligated into pGEM-T easy cloning vector by TA cloning. After digestion with restrictive enzyme and sequencing, the correct DNA fragment was inserted into inducible proeukaryotie expression vector pET-32a （ ＋ ）. The competent BL21 （DE3） E. coli was transformed, and then cultured and induced with IPTG. The expressed HBeBP4A was analyzed and confirmed with sodium dodeeyl sulfate-polyaerylamide gel eleetrophoresis （SDS-PAGE） and Western blotting hybridization. Results： The spliced DNA fragment of HBeBP4A was successfully amplified by RT-PCR, and its expression vector was constructed. After transformation with pET-32a （ ＋ ） -HBeBP4A and induction with IPTG, recombinant HBeBP4A was expressed and confirmed by SDS-PAGE and Western blotting. Conclusion： A novel genebinding protein 4 pliced variant HBeBP4A of hepatitis B virus e antigen has been recognized, and its recombinant prokaryotie expression vector pET-32a （ ＋ ） -HBeBP4A has been constructed. The recombinant HBeBP4A gene can be expressed in prokaryotic expression system of E. coll.
出处
《中西医结合肝病杂志》
CAS
2007年第6期348-351,371,共5页
Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases
基金
国家自然科学基金资助项目(No.30371288)
北京市自然科学基金项目(No.5042024)
关键词
E抗原
结合蛋白
剪切体
生物信息学
基因克隆
原核表达
e antigen
binding protein
spliced variant
bioinformatics
gene cloning
prokaryotic expression
