CAJ | 학술논문

目的通过对艰难梭菌毒素B受体结合区进行基因克隆、表达,获得目的蛋白,为建立快速检测艰难梭菌的方法奠定基础.方法复苏艰难梭菌,提取基因组DNA,PCR扩增目的基因,回收后与pGEM-T连接进行TA克隆,通过PCR、酶切及基因测序鉴定pGEM-T-CDB3.酶切pGEM-T-CDB3和pGEX-4T-1,回收CDB3和pGEX-4T-1,连接后转化大肠埃希菌BL21(DE3),构建重组质粒pGEX-4T-1-CDB3,然后应用IPTG诱导目的蛋白表达,通过聚丙烯酰胺凝胶电泳和Western blot鉴定目的蛋白大小和特异性.结果经PCR扩增获得了艰难梭菌毒素B受体结合区(CDB3)基因全长片段1851 bp.克隆质粒pGEM-T与CDB3重组获得了质粒pGEM-T-CDB3,片段长为4800 bp左右.构建了重组表达质粒pGEX-4T-1-CDB3,目的蛋白经诱导后获得表达.结论原核蛋白表达载体pGEX-4T-1-CDB3构建成功,并大量表达出目的蛋白,将对艰难梭菌毒素B的快速检测和疫苗的研制奠定良好基础.(中华检验医学杂志,2013,36:324-328)
목적 통과대간난사균독소B수체결합구진행기인극륭、표체,획득목적단백,위건립쾌속검측간난사균적방법전정기출.방법 복소간난사균,제취기인조DNA,PCR확증목적기인,회수후여pGEM-T련접진행TA극륭,통과PCR、매절급기인측서감정pGEM-T-CDB3.매절pGEM-T-CDB3화pGEX-4T-1,회수CDB3화pGEX-4T-1,련접후전화대장애희균BL21(DE3),구건중조질립pGEX-4T-1-CDB3,연후응용IPTG유도목적단백표체,통과취병희선알응효전영화Western blot감정목적단백대소화특이성.결과 경PCR확증획득료간난사균독소B수체결합구(CDB3)기인전장편단1851 bp.극륭질립pGEM-T여CDB3중조획득료질립pGEM-T-CDB3,편단장위4800 bp좌우.구건료중조표체질립pGEX-4T-1-CDB3,목적단백경유도후획득표체.결론 원핵단백표체재체pGEX-4T-1-CDB3구건성공,병대량표체출목적단백,장대간난사균독소B적쾌속검측화역묘적연제전정량호기출.(중화검험의학잡지,2013,36:324-328)
Objective To express receptor-binding region of Clostridium difficile toxin B by gene cloning,providing foundation for establishment of a rapid method to detect Clostridium difficile.Methods Recover Clostridium difficile,extract genomic DNA and amplify the targeted gene by PCR.The recovered PCR product was linked to pGEM-T by TA cloning.pGEM-T-CDB3 was identified by PCR,restriction enzyme cleavage and gene sequencing.The plasmids pGEM-T-CDB3 and pGEX-4T-1 were digested.The target gene CDB3 and pGEX-4T-1 were recovered,ligated and transformed into E.coli BL21 (DE3) to construct the recombinant plasmid.The expression of target protein was induced by IPTG,while the molecular weight and specificity of the protein were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results The PCR product of Clostridium difficile toxins B receptor-binding domain (CDB3) was about 1851 bp.Plasmid pGEM-T-CDB3 was about 4800 bp,obtained by recombinatiom the cloned vector pGEM-T with CDB3.The recombinant expression plasmid pGEX-4T-1-CDB3 was constructed and the target protein was expressed after induction.Conclusion The prokaryotic expression vector pGEX-4T-1-CDB3 is constructed successfully and the target protein is expressed greatly,which will lay a good foundation for rapid detection of Clostridium difficile toxin B and development of a vaccine.(Chin J Lab Med,2013,36:324-328)