羊巴贝斯虫未定种trap基因的克隆表达及反应原性分析

Cloning,expression and immunoreactivity analysis on Babesiasp.(Xinjiang)trap gene

通过克隆trap基因，在原核表达系统中进行表达并纯化，Westernblot鉴定TRAP蛋白反应原性，应用生物信息学分析软件分析验证该基因／蛋白的结构。以羊巴贝斯虫未定种新疆株gDNA和cDNA为模板扩增trap基因全长；并构建pET-30a-trap原核表达载体。将构建成功的表达载体转入BL21（DE3）pLysS进行诱导表达；纯化可溶性的rBspTRAP，Westernblot分析其反应原性，并应用生物信息学分析软件分析验证该基因／蛋白的结构。结果显示：获得的traP基因全长为2338bp，开放阅读框大小为1944bp，具有5个内含子和6个外显子。氨基酸序列分析显示1～26位氨基酸为信号肽序列，45～201位和240～288住氨基酸分别为TRAP蛋白家族的vWA和TSPl结构域。Westernblot结果显示羊巴贝斯虫未定种新疆／敦煌株阳性血清可特异性识别rBspTRAP，该重组蛋白与莫氏巴贝斯虫（临潭株、天祝株、河北株、宁县株）阳性血清及尤氏泰勒虫阳性血清无交叉反应。本试验成功克隆trap基因并表达，该基因可以作为免疫学诊断用候选抗原，为以后建立鉴别诊断莫氏巴贝斯虫和羊巴贝斯虫感染的免疫学诊断方法奠定基础。

The study was conducted to clone and express trap gene of Babesia sp. （Xinjiang）,analyze and verify their structures via bioinformatics softwares. The complete length of trap gene was amplified from genomic DNA and cDNA of Babesia sp. （Xinjiang）, then a prokaryotic expression vector, pET-30a-trap, was constructed and transformed into Escherichia coli BL21 （DE3） pLysS to induce protein expression. Soluble rBspTRAP was purified and then its immunoreactivity was determined By Western blot. Analysis results using bioinformatics softwares showed that the trap gene is 2 338 base pairs, contained 5 introns and 6 exons with an open reading frame（ORF） of 1 944 bp. The 1-23 animo acids was a signal peptide sequence and these of 45-201 and 240-288 were vWA and TSP1 motifs of TRAP protein family,respectively. The rBspTRAP could be specifically recognized by positive sera from sheep infected by Babesia sp. Xingjiang/Dunhuang. No cross-reactions were present on the sera of B. motasi Lintan/Tianzhu/Hebei/Ningxian and Theileria uilenbergi . This study successfully cloned and expressed trap gene, and rBspTRAP has the potetials for considering as a candidate of antigen using in sero-diagnostic methods. A foundation was established for developing immulogical diagnostic methods for differentiating the infection of Babesia. sp and B. motasi in future.