旋毛虫ES抗原基因的克隆和序列分析

Molecular cloning and sequencing of Trichinella spiralis ES antigen

目的 完成旋毛虫ES抗原 (Excretory -secretoryAntigen)中 4 3kDa分泌性糖蛋白基因和 5 3kDa分泌性抗原基因的克隆和测序 ,并与已发表的该两种基因的相关序列比较、分析。方法 通过RT -PCR ,从旋毛虫幼虫总RNA中扩增得到特异性片段 ,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。 结果 RT -PCR扩增得到 4 3kDa分泌性糖蛋白基因和 5 3kDa分泌性抗原基因 ,序列分析表明 ,其核苷酸序列与已发表的 4 3kDa分泌性糖蛋白基因和 5 3kDa分泌性抗原的同源性均为 99%。结论 成功提取了旋毛虫幼虫的总RNA ,并用PT -PCR方法克隆了 4 3kDa分泌性糖蛋白基因和 5 3kDa分泌性抗原基因 ,这为两基因的表达及在医学、兽医学中应用研究奠定了基础。

Aim To complete the cloning and sequencing of Trichinella spiralis ES antigen (Excretory secretory Antigen)43kDa secreted glycoprotein and 53kDa excretory secretory antigen,and compare both of the sequences with those reported by the other authors Methods Two pairs of primers were designed according to gene sequence of the published 43kDa secreted glycoprotein and 53kDa excretory secretory antigen in the database Using RT PCR to amplify the specific gene fragment from the total RNA extracted from the Trichinella spiralis larva In addition,different PCR products were linked to the T vectors respectively and the recombinant plasmids were verified by sequencing The sequence results were analysized with BLAST software Result The 43kDa secreted glycoprotein and 53kDa excretory secretory antigen gene have been successfully cloned using the RT PCR Sequence analysis showed that the two genes were consistant to the 43kDa and 53kDa gene sequences in the database After BLAST analysis the genes obtained were 99% identical to the 43kDa and 53kDa gene in the database Conclusion We extracted high quality total RNA from the Trichinella spiralis larva and cloned the 43kDa and 53kDa genes using PCR The present work have laid a foundation for the future 43kDa and 53kDa genes expression and the related immunology research