摘要
[目的]研究牛瑟氏泰勒虫P23表面蛋白基因的克隆及原核表达。[方法]采用PCR方法扩增牛瑟氏泰勒虫中国延边株P23基因片段,将扩增产物克隆入pMD18-T载体构建重组质粒pMD18-P23,经PCR、双酶切鉴定后测序;将目的基因片段亚克隆入表达载体pGEX-4T-1构建重组表达质粒pGEX-4T-P23,转化宿主菌BL21获得重组菌。通过对诱导条件的优化,根据SDS-PAGE确定表达蛋白的最佳表达条件;Western-blotting检测表达蛋白的反应原性。[结果]所克隆的牛瑟氏泰勒虫P23基因片段长507 bp,与牛瑟氏泰勒虫日本株P23基因的核苷酸同源性达99.4%,表达的融合蛋白大小约为46 ku;诱导时机以接种培养后2 h为最佳,诱导时间以6 h为最佳,诱导温度以34℃为最佳,0.008-1.000 mmol/L的IPTG对表达量的影响不大。Western blotting检测表明该蛋白具有较好的抗原性。[结论]为牛瑟氏泰勒虫病的免疫学诊断和预防等研究奠定了基础。
[Objective]The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti.[Method] A pair of specific primers were designed according to the sequence of P23 surface protein of Theileria sergenti（D84447）.The P23 gene was amplified by PCR from genomic DNA of Theileria sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23.Positive clones were identified by PCR screening and restriction digestion.A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E.coli BL21.After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting.[Result]The result showed that the cloned gene had a total length of 507 bp.Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank（D84447） the expressed fusion protein was 46 ku in molecular mass;Induction opportunity of zhours after culture inoculation was the best as well,the induction time of 6 hours was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1.000 mmol/L had little effect on the expression.Western-blotting indicated that recombinant protein was recognized by specific antibody.[Conclusion]This study will lay a foundation for further research on the prevention and diagnose of Theileria sergenti.
出处
《安徽农业科学》
CAS
北大核心
2010年第16期8462-8465,8483,共5页
Journal of Anhui Agricultural Sciences
基金
吉林省科技发展计划项目(20050703-4)
