pGEX-4T-1-cMC4R原核表达载体的构建与表达

Construction and Expression of Prokaryotic Expression Vector pGEX-4T-1-cMC4R

[目的]构建犬MC4R基因编码区的融合表达载体并在大肠杆菌中诱导表达融合蛋白。[方法]以Beagle犬基因组DNA为模板,经PCR技术扩增目的片段,利用LP Recco PCR克隆技术将目的片段直接重组到原核表达载体pGEX-4T-1上,转化到E.coliDH5α,筛选阳性克隆,BamHI、XhoI酶切鉴定,DNA测序检测插入序列的正确性。将测序正确的重组表达质粒pGEX-4T-1-cMC4R转化到E.co-liBL21内,经IPTG诱导后,利用SDS-PAGE检测犬MC4R蛋白的表达。[结果]成功构建重组表达质粒pGEX-4T-1-cMC4R,经DNA测序证实插入序列与设计基本一致,只有777位碱基T变成了C,这是由于犬MC4R编码区存在的多态性引起,对表达的犬MC4R蛋白序列无影响。大肠杆菌诱导后表达出犬MC4R融合性蛋白。[结论]成功构建犬MC4R原核表达载体,此重组体能在E.coliBL21内表达犬MC4R融合蛋白,为进一步获取犬MC4R的单克隆抗体奠定物质基础,也为研究犬MC4R蛋白的结构和生理功能提供帮助。

[ Objective] The aim was to construct an expression vector pGEX-4T-1-cMC4R and to express the fusion protein GST-cMC4R. [ Method] Using the canis MC4R DNA as template, the specific primers were designed. After PCR amplification, product was cloned into pGEX-4T-1 vector by LP Recco PCR cloning technique, the recombinant pGEX-4T-1 -cMC4R was transferred to E. coli DH5α, then, it was identified with double restriction enzymes digestion analysis and DNA sequencing. The recombinant pGEX-4T-1-cMC4R which was identical with GeneBank was transformed into E. coli BL21. The expression of canis MC4R protein was analyzed by SDS-PAGE after IFFG induction. [ Result] The construction of prokaryotic plasmid pGEX-4T-1-cMC4R was achieved. Canis MC4R sequence was mainly identical with the Gen6Bank. Due to the polymorphism of the MC4R gene in the dogs, T changed into C in the site of 777. E. coli BL21 expressed the fusion protein of canis MC4R. [ Conclusion] The construction and the expression of prokaryotic plasmid pGEX-4T-1-cMC4R was achieved successfully.