旋毛虫p49基因的克隆、序列分析及表达

目的 获得旋毛虫排泄分泌抗原(excretory antigen,ES)p49基因的克隆、测序及表达。 方法 通过RT-PCR,从旋毛虫幼虫总RNA中扩增得到特异片段,利用TA克隆将PCR产物克隆入pUC-T载体中并进行测序及同源性比较,并定向克隆到表达载体pEG-4T-3中,转化感受态细胞,诱导表达。 结果 RT-PCR扩增得到p49基因,其核苷酸序列与已发表的p49基因序列一致;BLAST分析表明其与旋毛虫p49基因、43 KDa分泌性糖蛋白同源性均为99％。经SDS-聚丙烯酰胺凝胶电泳,重组蛋白诱导表达在67 KDa处有一新蛋白带。 结论 提取旋毛虫幼虫总RNA,用RT-PCR方法克隆并表达了p49基因。

Cloning, Sequencing and Expression of Trichinella spiralis p49 Gene

Objective To conduct cloning, sequencing and expression of Trichinella spiralis ES antigen p49 gene. Methods RT-PCR was used to amplify the specific gene fragment from the total RNA of Trichinella spirais larvae. The PCR product was ligated to the T-vector and the recombinant plasmid was verified by sequencing. T-p49 and pGE-4T-3 were treated by both BamHI and XhoI. The ligation reaction was catalyzed by T4 DNA ligase. Results The p49 gene was cloned by using RT-PCR. Sequence analysis showed that the p49 gene obtained was consistent to the p49 sequence reported in the database. The expressed protein was shown as a new band at SDS-PAGE. BLAST analysis demonstrated that this p49 gene was 99% identical to the p49 gene reported and to the 43 kDa secreted glycoprotein gene in the database. Conclusion p49 gene from Trichinella spiralis larvae was cloned, sequenced and expressed.