镰形扇头蜱肌钙蛋白Ⅰ基因的克隆及其在大肠杆菌中的表达

CLONING AND EXPRESSION OF RHIPICEPHALUS HAEMAPHYSALOIDES TROPONIN Ⅰ GENE IN ESCHERICHIA.COLI

目的克隆镰形扇头蜱肌钙蛋白Ⅰ(TnI)的cDNA并进行表达及鉴定。方法用RT-PCR方法从镰形扇头蜱总RNA中克隆编码肌钙蛋白Ⅰ的cDNA片段,将该cDNA连接到pET-32a(+)表达载体,并转入宿主菌BL21(DE3),经IPTG诱导表达,并对表达产物进行Western blot分析。结果获得镰形扇头蜱的eDNA,并在大肠杆菌中以包含体的形式高效表达,Westem blot分析表明,抗TnI重组蛋白兔血清可以识别蜱体内的TnI,并在22 kDa和36 kDa之间有两条带。结论蜱体内可能同时存在两个大小不等的TnI,为进一步的研究打下基础。

Objective The cloned cDNA of Rhipicephalus haemaphysaloides troponin Ⅰ（TnⅠ） was inserted into pET32a（ ＋ ） to construct an prokaryotic expression vector. This recombinant was then transformed into Escherichia, coli in order to obtain TnⅠ protein as the foundation for further study. Methods The cDNA fragment was amplified by RT-PCR from R. haemaphysaloides total RNA and inserted into pET32a（ ＋ ） to construct a prokaryotic expressing vector. The recombinant was cloned into BL21 （DE3） bacteria and induced by IPTG to express in it, then the result of expression was detected by Western blot. Results The results revealed that TnⅠ cDNA were cloned successfully and this cDNA could be expressed in E. coli with high level. Western blot analysis suggested that the rabbit anti-TnⅠ serum can recognize corresponding protein in R. haemaphysaloides, and there were two bands between 22 kDa to 36 kDa. Conclusion Native TnⅠ may have two isoforms at the same time in tick