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目的:课题组前期研究已经筛选出日本血吸虫肺期童虫的差异基因-生长激素诱导的跨膜蛋白(GHITM)基因,其可能在日本血吸虫的生长发育中起关键作用。本文克隆、表达GHITM基因,以便进一步探索其在血吸虫生长发育过程中的作用。方法:根据GHITM基因的序列,分别设计含有酶切位点的GHITM基因引物,以日本血吸虫成虫的总RNA为模板进行RT-PCR,PCR产物提纯后使用EcoR I和XhoI双酶切,然后与使用同样酶双酶切的pET-28a-c质粒连接并转化DH5α宿主菌,使用卡那霉素筛选阳性克隆后测序。
OBJECTIVE: In the previous study, we have screened out the GHITM gene, a differential gene of Schistosoma japonicum, which may play a key role in the growth and development of Schistosoma japonicum. In this paper, GHITM gene was cloned and expressed to further explore its role in the growth and development of schistosomiasis. Methods: According to the sequence of GHITM gene, GHITM gene primers containing restriction enzyme sites were designed respectively. RT-PCR was performed using the total RNA of Schistosoma japonicum as a template. The PCR products were purified and double-digested with EcoR I and XhoI. The same enzyme digested pET-28a-c plasmid was ligated and transformed DH5α host bacteria, kanamycin screening positive clones after sequencing.