克隆与表达产甲酸草酸杆菌代谢基因Frc及临床意义  

Clinical significance of cloning and expression of frc of oxalobacter formigenes

作　　者：赖德辉[1] 李逊[1] 雷鸣[2] 曾国华[2] 袁坚[2] 吴文起[2] 何永忠[1] 何朝晖[2] 

机构地区：[1]广州医学院港湾医院泌尿外科,510700 [2]广州医学院第一附属医院泌尿外科,510230

出　　处：《中华腔镜泌尿外科杂志（电子版）》2010年第3期53-56,共4页Chinese Journal of Endourology(Electronic Edition)

基　　金：国家自然基金资助项目(30801140);广东省科技厅资助项目(2009B080701026);广州市卫生局资助项目(2008-YB-153)

摘　　要：目的研究产甲酸草酸杆菌草酸代谢基因Frc转化大肠杆菌BL21后稳定表达代谢草酸相关性酶--甲酰辅酶A转移酶(FCoAT)对草酸的降解效能。方法成功培养产甲酸草酸杆菌后,采用PCR方法从产甲酸草酸基因组中获得Frc基因,克隆到pMDTM19-T载体上进行测序,得到正确的基因片段。经双酶切将目的基因片段插入到原核表达载体PGEX-4T-2上,测序正确后,转化大肠杆菌BL-21,IPTG诱导表达GST-FCoAT融合蛋白,表达产物行western-blot鉴定分析。结果重组克隆载体pMDTM19-Frc经测序鉴定序列正确。成功构建融合原核表达质粒PGEX-4T-2-Frc,大肠杆菌BL21成为载体,并稳定表达可溶性融合蛋白GST-FCoAT的同时获得代谢草酸潜能。结论克隆产甲酸草酸杆菌Frc基因,成功构建PGEX-4T-2-Frc载体,并转化大肠杆菌BL21,能稳定表达可溶性融合蛋白GST-FCoAT,具有代谢草酸潜能,为肠道细菌获得代谢草酸潜能,减少胃肠道内草酸的吸收,降低尿液中草酸含量和临床防治草酸结石的研究奠定理论基础。Objective To study the potential of degrading oxalate for oxalate-degradation gene FRC of Oxalobacter formigenes transforming into E.coli BL21 and expressing oxalate-degradation associated enzyme-FCoAT（Formyl-CoA Transferase）.Methods Based on successful cultivation of oxalobacter formigenes,the oxalate-degradation gene FRC was amplified from the bacterial genome by PCR,and was cloned into vector pMDTM 19-T.After verified by sequencing,it was cut with restriction endonucleases BamH I and EcoR I,and then the Frc was extracted and inserted into the prokaryotic expression vector PGEX-4T-2 and verified by sequencing.The recombinant expression plasmid PGEX-4T-2-Frc was transformed into E.coli BL21,and induced to express the GST-FCoAT fu-sion protein with IPTG.Expressed protein was identified and analyzed by western-blot.Results Sequence analysis proved that the FRC was correct and prokaryotic expression plasmid PGEX-4T-2-FRC was constructed successfully.E.coli BL21 can stably express the GST-FCoAT fusion protein and obtain the potential of degrading oxalate.Conclusions The gene Frc was successfully cloned into the vector PGEX-4T-2-Frc and transformed into E.coli BL21.The expression of soluble fusion protein GST-FCoAT can obtain the potential of degrade oxalate for intestinal bacterial,which can reduce the absorption of oxalate in the gastrointestinal tract and decrease urinary oxalate concentration to prevent and treat oxalate stone.

关 键 词：产甲酸草酸杆菌 FRC 基因克隆 大肠杆菌BL-21 

分 类 号：R378[医药卫生—病原生物学]