重组鼠疫汉坦钩端多抗原位点同源合成基因的表达及活性鉴定  

Expression and Preliminary Activity Identification of Several Linked Antigen Epitopes of Consensce Genes of Yersinia Pestis,Hantavirus and Leptospira

作　　者：浦昀[1,2] 齐燕飞[1] 徐国良[3] 徐卉[3] 张士尧[1] 孟日增[1,2] 宋秀玲[1] 杨怀宁[2] 李娟[1] 

机构地区：[1]吉林大学公共卫生学院,长春130021 [2]吉林省出入境检验检疫局,长春130062 [3]吉林大学第一医院心血管中心,长春130021

出　　处：《高等学校化学学报》2011年第1期84-87,共4页Chemical Journal of Chinese Universities

基　　金：国家"重大新药创制"科技重大专项项目(批准号:2009ZX09103-105);高等学校博士学科点专项科研基金(批准号:20090061120093);国家质量监督检验检疫总局科技计划项目(批准号:2009IK214);吉林大学研究生创新研究计划项目(批准号:20091028)资助

摘　　要：将人工合成的含鼠疫耶尔森氏菌的2个抗原位点、汉坦病毒的5个抗原位点和钩端螺旋体的5个抗原位点的串联基因片段克隆到原核表达载体pET-20b中,构建重组表达质粒pET-rYHL.转化E.coliBL21(DE3)后获得表达菌株.表达菌株经IPTG诱导后,可高效表达带组氨酸标签的以包涵体形式存在的融合蛋白.包涵体蛋白经尿素变性溶解、His Trap HP Kit柱纯化、SDS-PAGE及Western Blot分析研究,结果表明,在分子量40000处有一特异性蛋白条带,获得纯度达98%以上的蛋白.进一步ELISA试验表明,该蛋白与鼠疫、流行性出血热及钩端螺旋体病阳性血清均能较好地结合,而与其它鼠传疾病血清无交叉反应.The expression vector pET-rYHL was constructed by inserting the linked gene contained two antigen epitopes of F1 capsule antigen from yersinia pestis,five antigen epitopes of hantavirus and five antigen epitopes of leptospira into pET-20b,and was identified by digestion with restriction enzymes and sequence analysis.Then an expression strain was selected after transformation of the recombined plasmid into E.coli BL21（DE3）,fusion protein with His-tag was efficiently expressed in the form of inclusion body after IPTG induction.The inclusion body was washed,dissolved and purified by Ni2＋ chelate chromatography under denatured condition.SDS-PAGE and Western Blot analyses show that the fusion protein with a molecular weight of about 40000 was purified and the purity was up to 98%.The results of ELISA show specific reactions with Plague,Epidemic hemorrhagic fever and Leptospirosis positive sera,respectively,and no cross-reaction with other positive sera sample（salmonella） using the expression protein.

关 键 词：鼠疫 汉坦病毒 钩端螺旋体 合成基因 基因表达 活性鉴定 

分 类 号：Q75[生物学—分子生物学] O69[理学—化学]