新疆牛环形泰勒虫Tams1基因的克隆与表达  

Cloning and expression of Tams1 gene from the bovine Theileria annulata in Xinjiang

作　　者：简子健[1] 黄家雨[2] 马素贞[2] 袁江玲[2] 苏贵成[2] 苗中秋[2] 沈炯玉[2] 张兰江 李晓军 

机构地区：[1]南京农业大学动物医学院,南京江苏210095 [2]新疆农业大学动物医学学院,乌鲁木齐新疆830052 [3]新疆克拉玛依市乌尔禾乡政府,新疆克拉玛依834012 [4]新疆克拉玛依市乌尔禾乡兽医站,新疆克拉玛依834012

出　　处：《中国预防兽医学报》2008年第10期784-789,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金资助项目(3066141)

摘　　要：采用PCR方法扩增环形泰勒虫Tams1基因,将扩增产物与pUcm-T载体连接,重组质粒经PCR和双酶切鉴定、测序确认后被亚克隆于pGEX-4T-2表达载体中。构建的表达重组质粒经IPTG诱导表达后,进行SDS-PAGE、Western blot分析。结果显示,克隆的Tams1基因片段长846bp,为一个完整的开放阅读框,编码281个氨基酸,与GenBank中的Tams1基因核苷酸同源性在93.6%～99.8%之间;系统进化树分析表明新疆分离株与印度株、毛利塔尼亚株和北非共和国株进化途径一致,与苏丹株、巴林株进化途径相差较远;推测的蛋白质抗原决定簇预测表明,Tams1基因编码的氨基酸具有13个交叉的抗原决定簇;表达的融合蛋白为56ku,能够被环形泰勒虫多克隆血清所识别,表明该融合蛋白具有较好的反应原性和免疫原性,为进一步开展牛环形泰勒虫的分子生物学诊断、亚单位疫苗和核酸疫苗的研究奠定了可靠的基础。The Tamsl gene of bovine Theileria annulata Xinjiang strain was amplified by PCR and sequenced. The Tamsl gene was 846 bp in length with an ORF encoding 281 amino acids. It shared 93.6 %-99.8 % nucleotide homology with that published in GenBank. Phylogenetic tree analysis showed that the Xinjiang strain is closely related to other strains from India, Mauritania, Turkey, Tunisia. Analysis of antigenic determinants showed that it had 13 putative cross-antigenic determinants. The cloned sequence was sub-cloned into expression vector pGEX-4T-2 and resultant construct was transformed into E.coli cells and induced by IPTG. SDS-PAGE and Western blot showed that a fusion protein of 56 ku was expressed which could be recognized by antiserum against the bovine Theileria annulata. This result indicated the fusion protein possessed strong immunogenicity, which could be used for molecular diagnosis and development of the subunit and nucleic acid vaccines.

关 键 词：牛环形泰勒虫 Tams1基因 克隆 表达 融合蛋白 

分 类 号：S852.72[农业科学—基础兽医学]