细粒棘球蚴中国大陆株线粒体苹果酸脱氢酶基因的克隆和序列分析

Cloning and sequence analyzing of the mMDH gene from echinococcus granulosus of China

目的获得细粒棘球蚴线粒体苹果酸脱氢酶(mMDH)基因片段,进行序列分析,为研究包虫病疫苗候选基因奠定基础。方法从包虫病患者体内获取细粒棘球蚴原头蚴提取总RNA,根据国外细粒棘球蚴mMDH(线粒体苹果酸脱氢酶)基因的已知序列设计一对引物,采用RT-PCR技术扩增出细粒棘球蚴中国大陆株mMDH基因,将PCR产物纯化后,亚克隆到pGEM-T载体并构建成基因工程菌株后进行序列测定和分析。结果用RT-PCR成功扩增出细粒棘球蚴中国大陆株mMDH基因,测序表明该基因开放阅读框为1017bp,与已发表基因核苷酸序列相比,同源性为99%,理论蛋白编码的氨基酸序列同源性为99%。结论在国内首次获得细粒棘球蚴线粒体苹果酸脱氢酶(mMDH)基因序列,为进一步构建重组表达基因工程菌株打下良好的基础。

Objective To obtain and analyze the mMDH gene sequence, and lay bases for screening candidate antigen of Echinococcus granulosus. Methods Total RNA was extracted from protoscoles of cysts from humam origin. The specific primers were designed according to the published nucletid sequence in the Genbank database. The mMDH gene of Echinococcus granulosus was amplified by RT- PCR and cloned into pGEM-T vector for sequencing and analyzing. Results A cDNA sequence with an open reading frame of 1017bp has been amplified successfully by RT- FCR. Comparision of the DNA and amino acid sequence was deduced from cDNA with the published mMDH gene sequence of Echinococcus granulosus in the Genbank revealed 99 % identity. Conclusion mMDH gene was gained in China from protoscoles can be used as candidate antigene gene to develop vaccine and its biological function study.