柔嫩艾美耳球虫二氢乳清酸脱氢酶基因注释、克隆与原核表达

Annotation , Cloning and Expression of Dihydroorotate Dehydrogenase fromEimeria tenella

二氢乳清酸脱氢酶(DHODH)是催化嘧啶从头合成途径中的关键酶,由于顶复门原虫等低等生物与其宿主的DHODH在分子结构上的差异,可以作为研发新型抗顶复门原虫药物的靶标而成为研究的热点。本研究利用电子克隆策略,以疟原虫DHODH氨基酸基因序列为信息探针,搜索球虫基因库(www.sanger.ac.uk/projects/E-tenella/),拼接注释了柔嫩艾美耳球虫(Eimeria tenella)DHODH的基因序列,以E.tenella广东株第2代裂殖子总RNA为模板,利用RT-PCR技术扩增获得EtDHODH ORF基因序列。构建重组表达载体pColdⅠ-EtDHODH,进行原核表达。测序结果显示,EtDHODH的基因大小为1 254bp,SDS-PAGE鉴定结果表明,目的蛋白约45ku。本研究重组表达并纯化了EtDHODH基因,为建立以EtDHODH为靶点的抗球虫药物筛选奠定了基础。

Dihydroorotate dehydrogenase（DHODH）is a key enzyme in de novo pyrimidine biosynthesis,which is explored as a potential drug target of anti-apicomplexa dependent on the molecular structure is different between apicomplexan parasites and their hosts.In this study,according to the technology of in silico cloning,the gene sequence of E.tenella DHODH was predicted fromE.tenellagene datebase using Plasmodium falciparum DHODH gene sequence as a querying probe,which was amplified by RT-PCR from total RNA extracted from total RNA extracted from the second generation merozoite of E.tenella（Guangdong strain）.The recombinant plasmid pColdⅠ-EtDHODH was constructed,transformed into E.coli Rosseta（DE3）,and induced with IPTG.The results indicated that the E.tenella DHODH ORF was 1 254bp in full length,and the molecular weight was about 45ku.In brief,the E.tenella DHODH recombinant protein was expressed and purified successfully,which provided the foundation for further development of the novel anti-coccidian drugs against E.tenella.