阴道毛滴虫14-3-3基因的克隆、表达及鉴定  

Cloning,expression and identification of the 14-3-3 gene of Trichomonas vaginalis

作　　者：刘畅[1] 丁鹤[1] 刘鑫[1] 宫鹏涛[1] 李建华[1] 李淑红[1] 李赫[1] 张国才[1] 杨举[1] 张西臣[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国病原生物学杂志》2012年第5期349-351,共3页Journal of Pathogen Biology

基　　金："艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(No.2008ZX10401-B)

摘　　要：目的克隆阴道毛滴虫14-3-3基因并进行原核表达。方法根据阴道毛滴虫14-3-3基因序列设计特异性引物,以阴道毛滴虫cDNA为模板通过PCR扩增获得目的片段,与pMD-18-T连接,构建克隆载体pMD-Tv-14-3-3,经双酶切后回收目的片段,进行测序鉴定,然后与表达载体pGEX-T连接,构建原核表达载体pGEX-T-Tv-14-3-3,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过SDS-PAGE及Western blot鉴定表达产物。结果成功构建了阴道毛滴虫14-3-3基因原核表达载体pGEX-T-Tv-14-3-3;SDS-PAGE电泳检测显示,在IPTG诱导下,阳性菌高效表达分子质量单位为27ku的蛋白质;Western blot显示表达产物可被抗阴道毛滴虫多克隆血清识别。结论成功构建了阴道毛滴虫14-3-3基因原核表达载体,并在大肠埃希菌BL21(DE3)中高效表达。Objective To clone and express 14 3-3 gene of Trichomonas vaginalis. Methods Special primers were de-signed on the hasis of the reported T. vaginalis 14-3-3 gene. The 14-3-3 gene was amplified by PCR from the total DNA of T. vaginalis and was cloned into pMD-18-T to construct pMD-14-3-3. The plasmid pMD-14-3 3 was then digested with restriction ribozymes and subcloned into the prokaryotic expression plasmid pGEX-T to construct pGEX-T Tv-14-3- 3. It was then expressed in E. coli BL21 （DE3） induced with IPTG. The fusion product was identified by SDS-PAGE and Western blot. Results A prokaryotic expression vector of the 14-3-3 gene was constructed and expressed in Esche-riehia, coll. Induced with IPTG, the expressed recombinant protein was detected as a band of 27 ku by SDS-PAGE. A special reaction band to anti-14-3-3 sera was observed in Western blot. Conclusion The fusion protein of the 14-3-3 gene was successfully expressed in prokaryotic ceils.

关 键 词：阴道毛滴虫 14-3-3基因 克隆 原核表达 

分 类 号：R382.21[医药卫生—医学寄生虫学]