摘要
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/G192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10 ku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。
Escherichia coil heat-labile enterotoxin （LT） is a potent mucosal immunogen and adjuvant in animal models. To inactivate or reduced the toxic activity of E. coli LT, the recombinant plasmid of pLTR72/G192 was constructed with two point mutations of R^72/G^192 in E. coli LT gene generated by PCR and SOE PCR, and the recombinant protein of LTR^72/G^192 was expressed in E. coli by IPTG induction. SDS-PAGE analysis showed that the subunits of LTA and LTB were 33.0 ku and 13.0 ku, respectively. The recombinant protein was reacted with anti-His-tag antibodies in western blot detection. ADP-ribosyltransferase activity and the toxicity assay in Patent-mouse test showed that the enzyme activity and toxicity of the LTR^72/G^192 was significantly decreased compared to wild-type LT. Furthermore, the ELISA results showed that the titers of IgG and slgA was significant higher in chickens immunized with LTR^72/G^192 adjuvanted Newcastle vaccine via intranasally inoculation than that of Newcastle vaccine alone.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2012年第9期697-701,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
上海市科技兴农重点攻关项目[2005攻字(10-1)]
