从新疆株细粒棘球绦虫(Echinococcus granulosus,Eg)原头蚴中克隆胆汁酸钠协同转运蛋白(sodium-bile acid co-transporter,EgSBACT)基因,进行序列测定和生物信息学分析。首先设计EgSBACT 基因特异性引物,用RT 及PCR 方法从新疆株细粒棘球绦虫原头蚴提取总RNA,将其反转录成cDNA 后扩增EgSBACT 基因并将其克隆至原核表达载体pET30a 中,测序鉴定序列并进行生物信息学分析。结果显示,RT-PCR 扩增出一长度约650 bp 的基因,测序显示其长度为654 bp,序列与Gen-bank 公布的EUB59978.1 完全一致,其编码217 个氨基酸,预测其等电点为9.28。同源性分析发现,EgSBACT 蛋白序列与中华枝睾吸虫SBACT(GenBank:GAA57409.1)同源性最高,达到43%。进化树分析表明,细粒棘球绦虫的SBACT 蛋白与吸虫纲的SBACT 相聚集,在进化树上处于一枝,而与其他物种亲缘性较远。由此表明,本研究成功克隆出细粒棘球绦虫EgSBACT 基因。
Sodium-bile acid cotransporter (EgSBACT) functions in the sodium-bile acid transportation in Echinococcus granulosus (Eg) Protoscolex and the sequence is unknown. The specific primers for EgSBACT mRNA are designed and the EgSBACT gene is amplified by RT-PCR from Eg protoscolex total RNA. Then the EgSBACT gene fragment is cloned into pET30a vector which is a prokaryotic expression vector for sequencing and analyzing by bioinformatics method. The PCR products of EgSBACT is about 650 bp whose length is 654 bp and encodes a protein containing 217 amino acids and the pI of this protein is predicted as 9.28. Homology analysis shows that there is 43% of similarity between EgSBACT and the trematode SBACT (GenBank: GAA57409.1). Phylogenetic analysis shows that EgSBACT is clustered with Fluke, and has a lower homology to other species. EgSBACT gene is cloned from protoscolex of Echinococcus granulosus in Xinjiang, which lays a foundation for further study of EgSBACT function in the development and differentiation of PSCs into adult worms. This research provides a way to express the EgSBACT protein in vitro and taking advantage of this protein further research can reveal the exact functions of EgSBACT in the sodium-bile acid transportation in Echinococcus granulosus.
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