CAJ | 학술논문

目的构建结核分枝杆菌(MTB) Rv0073基因原核表达载体并进行表达和纯化.方法以MTB H37Rv基因组DNA为模板,采用聚合酶链反应(PCR)扩增目的基因片段,构建原核表达载体pET26b-Rv0073,经测序确定无误后转化至大肠杆菌(E.coli)感受态细胞BL21中.用聚丙烯酰氨凝胶电泳(SDS-PAGE)方法检测重组蛋白表达,检测异丙基-β-D-硫代半乳糖苷(IPTG)诱导不同时间、不同温度条件下重组蛋白表达量.采用His镍磁珠进行外源蛋白小量纯化.结果成功构建重组表达质粒,重组蛋白经IPTG诱导后,2h开始明显表达且表达量无时间依赖性,在不同温度诱导下,重组蛋白的表达量随温度的增高而减少.重组蛋白以包涵体形式存在,经His镍磁珠纯化后获得重组蛋白.结论成功构建并表达Rv0073蛋白,为后续Rv0073的大量纯化及其功能研究奠定了基础.
목적 구건결핵분지간균(MTB) Rv0073기인원핵표체재체병진행표체화순화.방법 이MTB H37Rv기인조DNA위모판,채용취합매련반응(PCR)확증목적기인편단,구건원핵표체재체pET26b-Rv0073,경측서학정무오후전화지대장간균(E.coli)감수태세포BL21중.용취병희선안응효전영(SDS-PAGE)방법검측중조단백표체,검측이병기-β-D-류대반유당감(IPTG)유도불동시간、불동온도조건하중조단백표체량.채용His얼자주진행외원단백소량순화.결과 성공구건중조표체질립,중조단백경IPTG유도후,2h개시명현표체차표체량무시간의뢰성,재불동온도유도하,중조단백적표체량수온도적증고이감소.중조단백이포함체형식존재,경His얼자주순화후획득중조단백.결론 성공구건병표체Rv0073단백,위후속Rv0073적대량순화급기공능연구전정료기출.
Objective To construct a prokaryotic expression vector carrying Mycobacterium tuberculosis (MTB) Rv0073 gene and express the recombinant protein in Escherichia coli(E.coli).Methods The Rv0073 gene from MTB H37Rv strain was amplified by polymerase chain reaction (PCR) and cloned into the plasmid pET26b(+).Then,E.coli BL21 strain was transformed with the recombinant plasmid.The recombinant protein expression was induced by using Isopropyl β-D-1-thiogalactopyranoside (IPTG)at 25 ℃,28 ℃,30 ℃,34 ℃ and 37 ℃,respectively.The protein expression by IPTG induction for 2 h,4 h and 7 h was also examined.Finally,we purified the recombinant protein using the MagneHisTM Ni-Parties kit.Results We constructed a prokaryotic expression plasmid,pET26b-Rv0073.The Rv0073 protein expression in BL21 was successfully detected by SDS-PAGE.Induction temperature did not affect the expression.But induction time is related to the expression and the expression of recombinant protein decrease with increasing induction temperature.The recombinant protein formed inclusion bodies after expression.We could successfully purify the protein.Conclusion The expression plasmid of Rv0073 will become the basis for further production of Rv0073 and for the studying on the biological functions of the protein.