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Expression and bio-activity of Escherichia coli heat-labile enterotoxin mutant proteins
Author(s): 
Pages: 697-701
Year: Issue:  9
Journal: Chinese Journal of Preventive Veterinary Medicine

Keyword:  大肠杆菌不耐热肠毒素免疫原性ADP-核酸转移酶;
Abstract: 为使大肠杆菌不耐热肠毒素(LT)的毒性丧失或减弱的同时仍保留其较强的免疫原性,本实验通过PCR和重叠-延伸PCR扩增,制备了突变体LTR72/G192的基因片段,经酶切和测序结果表明构建的表达载体pLTR72/G 192阅读框架正确,而且相应位点氨基酸获得了替换.IPTG诱导表达目的蛋白经SDS-PAGE电泳检测,表达出突变体重组蛋白为约30 ku和10ku的两个蛋白带,与LTA、LTB亚基分子量相吻合.Western blot检测结果表明两个蛋白亚基均可与His抗体发生特异性反应.目的蛋白经纯化后进行ADP-核酸转移酶试验及Patent-mouse毒性试验检测其酶活性与毒性,突变重组蛋白与野生型LT相比其酶活性和毒性均明显降低.纯化的目的蛋白与鸡新城疫病毒弱毒疫苗联合一起经滴鼻免疫鸡,ELISA结果显示,突变体LTR72/G192能辅助新城疫疫苗在血清和黏膜中产生较高滴度的抗新城疫病毒的IgG和IgA.
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