刚地弓形虫LDH-Like MDHcDNA的克隆和序列分析  

Cloning and sequence analysis of the lactate dehydrogenase-like malate dehydrogenase cDNA from Toxoplasma gondii

作　　者：申川军[1] 何蔼[1] 郑小英[1] 余南[1] 郑斌[1] 郑焕钦[1] 李卓雅[1] 曹爱莲[1] 詹希美[1] 

机构地区：[1]中山大学基础医学院寄生虫学教研室,广州510089

出　　处：《中国人兽共患病杂志》2005年第6期461-466,490,共7页Chinese Journal of Zoonoses

基　　金：中山大学科研基金;中山大学基础医学院科研基金资助

摘　　要：目的获取弓形虫RH株速殖子苹果酸脱氢酶(MDH)的cDNA全长序列,并对推导翻译出的氨基酸(aa)序列进行分析。方法将NCBIGenBank中登录的弓形虫MDHEST序列进行比对、拼接和电子延伸,发现近5’末端的部分核酸序列缺失。基于分析,设计的上游、下游引物分别包含起始密码子ATG和终止密码子TGA(TGA→UGA),使经RTPCR扩增出的片段对应完整的CDS或者ORF。之后对MDHCDS推导翻译出的aa序列进行了保守功能域和同源性分析。结果本研究克隆的MDH的cDNA全长951bp,推导翻译出的蛋白质有316个aa组成,约35kDa,与预期大小接近。保守功能域分析表明,弓形虫MDH最接近类乳酸脱氢酶型(lactatedehydrogenaselike)MDH,其准确定名缩写为LDHlikeMDH,同时弓形虫MDH还具有其它脱氢酶类的特征。弓形虫MDH除与隐孢子虫和恶性疟原虫高度同源外,还与很多细菌等物种同源,但与人的同源性最低(22%)。弓形虫MDHcDNA序列在NCBIGenBank中登录号为AY650028,对应aa序列的登录号为AAT67462。结论本研究首次获得了弓形虫MDH基因的全长cDNA序列和对应的aa序列。MDH是三羧酸循环中的重要酶类,该酶活性的改变,将直接影响弓形虫的存活。弓形虫MDHaa序列与人MDH蛋白间的同源性很低,提示基于MDH蛋白作为药靶,经高通量技术筛选抗弓形虫药具有可行性。The malate dehydrogenase(MDH) cDNA from Toxoplasma gondii was cloned by RT-PCR using the primers designed on the basis of 11 ESTs of T.gondii MDH. The amplified single fragment was ligated with sequencing vector pMD18-T and the recombinant pMD18-T-MDHs were sequenced with identify of double restriction and PCR. Then the cDNA and the putative amino acids of MDH gene were analyzed bioinformatically. The results showed that the newly identified 951bp cDNA fragment in length just corresponded to an ORF or a complete CDS which coded 316 amino acids. The analysis data of conserved function domains and homology indicated that the cloned gene was clearly defined as belonging to the NADH-dependent LDH-like MDHs. T.gondii MDH also possessed partial characters from other dehydrogenases. There was very high homology between T. gondii and Cryptosporidium parvum, Plasmodium falciparum, as well as some bacteria. But T. gondii MDH had greatly low homology with human, the important intermediate host for T. gondii. In conclusion, T.gondii MDH cDNA was firstly cloned in the present study. The markedly low homology between T.gondii and human suggests that it might be feasible for choosing MDH as a drug target to screen anti-T. gondii drugs by means of high-through technology.

关 键 词：弓形虫 苹果酸脱氢酶 克隆 序列分析 

分 类 号：R382.5[医药卫生—医学寄生虫学]