驽巴贝斯虫截短HSP70基因克隆、表达及其生物信息学分析  

Cloning,Expression and Bioinformatic Analysis of the Truncated HSP70 Gene of Babesia caballi

作　　者：张梦圆[1] 张杨[1] 宋晶晶 肖培培 樊新丽[1] 李敏 吾力江 李才善 巴音查汗·盖力克 ZHANG Mengyuan;ZHANG Yang;SONG Jingjing;XIAO Peipei;FAN Xinli;LI Min;WU Lijiang;LI Caishan;GAILIKE Bayinchahan(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《中国动物传染病学报》2022年第3期173-180,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然基金地区科学基金项目(31660711);新疆维吾尔自治区自然科学基金项目(2016D01B027)。

摘　　要：为了对驽巴贝虫(Babesia caballi)HSP70基因进行原核表达获得生物信息学数据。本试验以新疆地方驽巴贝斯虫株截短HSP70基因组DNA为模板进行PCR扩增,序列比对分析,构建重组表达质粒HSP70-pET32a,经过IPTG诱导表达,用SDS-PAGE和Western blot鉴定。并用纯化后的蛋白免疫小鼠,制备多克隆抗体。经生物信息学分析,该蛋白属于碱性、稳定亲水性蛋白,有7个抗原位点。SDS-PAGE结果表明,重组蛋白大小为33 kDa,以包涵体蛋白的形式高效表达;Western blot结果表明,表达产物可被自然感染的马驽巴贝斯虫阳性血清识别,具有良好的免疫原性。经检测多克隆抗体效价可达1∶1 024 000。本试验将为进一步探究驽巴贝斯虫HSP70蛋白作为抗驽巴贝斯虫疫苗和免疫学诊断方法的候选抗原及其功能研究提供了参考依据。To obtain bioinformatics data for prokaryotic expression of Babesia caballi HSP70 gene,the truncated HSP70 genomic DNA of B.caballi from Xinjiang was used as a template for PCR amplification,sequence analysis and construction of recombinant expression plasmid HSP70-pET32a.The recombinant HSP70 protein was expressed in E.coli with induction of IPTG and characterized by SDS-PAGE and Western blot.Polyclonal antibodies were prepared by immunizing mice with purified proteins.According to bioinformatics analysis,this truncated HSP70 protein was an alkaline and stable hydrophilic protein with 7 antigen sites.SDS-PAGE results showed that the size of recombinant protein was 33 kDa,which was highly expressed in the form of inclusion body.Western blot results showed that the expressed product was recognized by the naturally infected positive serum of manabaeus.The titer of polyclonal antibodies reached to 1∶1 024 000.This study provided a reference for further research on B.caballi HSP70 protein as a candidate of anti-B.caballi vaccine and immunological diagnostic reagent.

关 键 词：驽巴贝斯虫 HSP70基因 原核表达 免疫原性 生物信息学 

分 类 号：S855.91[农业科学—临床兽医学]