摘要
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pGEx4T－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Westernblot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1851bp。克隆质粒pGEM—T与CDB3重组获得了质粒pGEM—T－CDB3，片段长为4800bp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。（中华捡验医学杂志，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 dcile. Methods Recover Clostridium difficile, extraet genornic 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. coil 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 diffcile toxin B and development of a vaccine. （ Chin J Lab Med,2013,36：324-328）
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2013年第4期324-328,共5页
Chinese Journal of Laboratory Medicine
基金
基金项目：中南大学研究生创新课题资助项目（201lssxt206）
湖南省卫生厅科研基金课题资助项目（2011-008）
