环形泰勒虫裂殖体表面蛋白基因高免疫原性区片段的克隆及其表达  

Cloning and expression of hyperimmunogenic region of surface protein gene of Theileria annulata schizonts

作　　者：党志胜[1] 罗建勋[1] 蒋锡仕[2] 黄孝玢[2] 白启[1] 殷宏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,甘肃兰州730046 [2]西南民族大学,四川成都610041

出　　处：《中国兽医科技》2004年第1期22-26,共5页Chinese Journal of Veterinary Science and Technology

基　　金：国家"8 6 3"项目 (2 0 0 1AA2 4 90 81) ;欧盟资助项目 (ICA4 CT 2 0 0 0 30 0 2 8) ;国家自然科学基金项目 (30 2 70 992 )

摘　　要：根据环形泰勒虫TaA12 72 1株裂殖体表面抗原 (TaSP)基因的序列设计了 1对引物 ,用PCR扩增出该基因长为 393bp的高免疫原性区片段 (SBxp)。将扩增产物连接到 pGEM TEasy载体上 ,转化入大肠埃希氏菌JM 10 9中进行克隆。随后将重组质粒 pGEM SBxp和表达载体 pGEX 4T 1分别以BamHⅠ、XhoⅠ酶切后 ,将酶切的目的片段SBxp亚克隆到原核表达载体中构建了重组表达载体 pGEX 4T 1 SBxp ,并将其转化到BL2 1宿主菌中。提取重组质粒经BamHⅠ、XhoⅠ酶切、PCR鉴定和测序确证后 ,阳性克隆用IPTG诱导表达。收集不同时间的菌液进行SDS PAGE电泳和Westernblotting检测。结果 ,SBxp基因在大肠埃希氏菌中成功表达 ,其表达产物为分子质量 4 3ku的融合蛋白 ,该产物能被牛环形泰勒虫阳性血清所识别。The total RNA was extracted from cell cultures of Theileria annulata schizonts and single chain cDNA was synthesized by reverse transcription using oligo(dT)_(18) as the primers. SBxp gene specific primers were designed by DNAstar software. A 393bp specific fragment was amplified by PCR and ligated into pGEM-T Easy vector. It was identified by restriction endonucleases, PCR and sequencing that this fragment contained the complete open reading frame of SBxp gene. After pGEM-SBxp recombinant vector was digested, SBxp gene was subcloned into the prokaryotic expression vector pGEX-4T-1, which was also digested with the same enzymes. Positive clones were identified by restriction endonuclease and PCR.The recombinant Escherichia coli was induced by IPTG and the cultures was collected at different times .The expressed products was tested by SDS-PAGE and Western blotting. The results showed that SBxp gene was expressed successfully in E.coli and the 43 ku fusion protein can be recognized by the positive serum of T.annulata.

关 键 词：环形泰勒虫 热带泰勒虫病 环形泰勒虫病 裂殖体 表面蛋白 基因 高免疫原性区 克隆 表达 重组质粒 

分 类 号：S852.723[农业科学—基础兽医学]