1
目 录
Abstract1
Key words1
引言2
1材料与方法2
1.1材料 2
1.2方法4
1.2.1旋毛虫肌肉幼虫收集4
1.2.2旋毛虫肌肉幼虫虫体RNA提取4
1.2.3旋毛虫肌肉幼虫cDNA合成5
1.2.4引物设计与比对5
1.2.5 PGRMC2基因扩增6
1.2.6 pMD19TTsPGRMC2重组质粒构建7
1.2.7 pET 32aTsPGRMC2重组表达载体的构建7
1.2.8 pET 32aTsPGRMC2重组载体的表达及分布鉴定8
1.2.9 镍离子亲和法纯化包涵体蛋白8
2.结果8
2.1基因扩增8
2.2完整ORF扩增12
2.3 重组表达质粒pET32aTsPGRMC2构建 13
2.4 重组蛋白在大肠杆菌中的诱导表达13
3讨论15
致谢16
参考文献17
旋毛虫膜相关孕酮受体组件2的基因克隆与表达
作者：耶赫亚江.麦麦提 指导老师：严若峰
摘 要：膜相关孕酮受体(membraneassociated progesterone receptor, mPGR)在哺乳动物生殖活动中发挥重要作用，而旋毛虫mPGR的功能尚不清楚。本实验根据旋毛虫mPGR组件2蛋白（Membraneassociated progesterone receptor component 2, PGRMC2）的基因序列，扩增了该蛋白基因的3’端序列，然后用5’RACE扩增出了其5’端，在此基础上获得了PGRMC2的ORF。序列分析结果表明PGRMC2的ORF大小为528bp，编码175个氨基酸，蛋白质理论大小为19.99kDa。将PGRMC2 ORF亚克隆到pET32a载体，构建了原核表达质粒pET32aTsPGRMC2，阳性克隆经IPTG诱导后获得了表达，重组融合蛋白大小为40kDa左右，主要以包涵体形式存在。利用Ni柱亲和层析法纯化了重组蛋白，为旋毛虫PGRM *51今日免费论文网|www.jxszl.com +Q: ^351916072# 
C2的功能研究奠定了基础。
Molecular cloning and expression of membraneassociated progesterone receptor component 2 (PGRMC2) from Trichinella spiralis
Student majoring in veterinary medicine: Yeheyajiang Maimaiti
Tutor: Yan Ruofeng
Abstract: Membraneassociated progesterone receptors (mPGR) play an important role in mammalian reproductive activity, while the function of Trichinella spiralis mPGR is unclear. The 3’ end of gene encoding membraneassociated progesterone receptor component 2 (PGRMC2) was amplified. And the 5 end of this gene was got by 5 RACE. Then the ORF of PGRMC2 was obtained by sequence analysis. Result showed that the PGRMC2 ORF was 528bp, encoding 175 amino acids, and the theoretical size of protein was 19.99kDa. The PGRMC2 ORF was subcloned into pET32a vector and the prokaryotic expression plasmid pET32aTsPGRMC2 was constructed. The positive clone was induced by IPTG, it was found that the recombinant protein sized as 40kDa was expressed in inclusion body. The recombinant protein was purified by Ni column affinity chromatography, which laid the foundation for the functional study of T. spiralis PGRMC2.
keywords: Trichinella spiralis; PGRMC2; Gene cloning ; Prokaryotic expression
旋毛虫（Trichinella spiralis, Tsp）是一种重要的人兽共患病病原，严重威胁食品卫生安全[13]。含有包囊的肉品未经充分煮熟而被食用后肌肉幼虫(muscle larvae, ML)释放出来，在小肠发育为成虫（adult worms, AD)，雌雄虫交配产出新生幼虫（newborn larvae, NBL)，NBL经淋巴循环和血液循环最终寄生于宿主横纹肌形成包囊[4]。目前，尚无旋毛虫商品化疫苗，寻找旋毛虫的药物靶标和疫苗候选分子对防治该病具有重要意义。

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