口蹄疫病毒3ABC基因截短体在毕赤酵母中的表达及鉴定  

Expression and Identification of FMDV 3ABC Truncated Gene in Pichia Pastoris

作　　者：郑敏[1] 金宁一[2] 李昌[2] 鲁会军[2] 马鸣潇[1] 沈国顺[1] 霍晓伟[1] 马海利[1] 陈晓月[1] 屈勇刚[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130062

出　　处：《高等学校化学学报》2008年第1期104-108,共5页Chemical Journal of Chinese Universities

基　　金：吉林省科技厅重大科研项目(批准号:20040202-1);国家“八六三”计划(批准号:2006AA0A204)资助

摘　　要：将长为525bp的口蹄疫病毒3ABC基因截短体克隆到毕赤酵母表达载体pPIC9K中,构建了重组表达质粒pPIC9K-3ABCt.用BglⅡ线性化后,电转化毕赤酵母菌GS115,经表型筛选,PCR鉴定,获得阳性重组菌(GS115/pPIC9K-3ABCt).然后进行诱导表达,通过SDS-PAGE和Western blot鉴定表达产物.结果表明,重组菌株成功分泌表达了分子量为40000,具有免疫反应活性,且呈二聚体形式的目的蛋白.在96h时表达量达到最高峰,占分泌总蛋白的18%,达到23.4mg/L.为进一步研制口蹄疫免疫和感染动物鉴别诊断试剂奠定了基础.Foot-and-mouth disease virus（FMDV） is an important pathogen worldwide; consequently, an important goal is the developments of diagnostic methods. To acquire an optimal diagnostic antigen allows one to distinguish vaccinated anionals from infected animals, a recombinant expression plasmid pPIC9K-3ABCt was constructed by inserting of FMDV 3ABC truncated gene（525 bp） into yeast expression vector pPIC 9K. Secondly, plasmid pPIC9K-3ABCt was linearized by BglII, and transformed into GS115 cells by electroporation. Positive clones were selected with MD/MM plates and confirmed by PCR and RT-PCR. Finally, expression product of 3ABCt was analyzed by SDS-PAGE and Western blot. The results show that the induced recombinant Pichia pastoris GS115/pPIC9K-3ABCt could secret 3ABC protein mainly in dimer form into culture supernatant, which had good immunoreactivity and antigen specificity and its molecular weight output was about 40000. Expression of 3 ABCt protein reached peek at 96 h after induction, maximum expression was accumulated up to 18% of the total supernatant protein, and production was about 23.4 mg/L.

关 键 词：口蹄疫病毒(FMDV) 3ABC基因 毕赤酵母 分泌表达 诊断抗原 

分 类 号：Q78[生物学—分子生物学] O629[理学—有机化学]