大肠埃希菌不耐热肠毒素双突变体的表达及活性研究

为了使大肠埃希菌不耐热肠毒素(LT)的毒性丧失或减弱的同时仍保留其较强的免疫原性，通过PCR和重叠—延伸PCR扩增突变体LTK⁶³/G¹⁹²基因片段， IPTG诱导表达目的蛋白，经Western免疫印迹检测目的蛋白，在HPLC系统上纯化目的蛋白，经ADP-核酸转移酶试验及Patent-mouse毒性试验检测其酶活性与毒性，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。结果表明：制备的突变体LTK⁶³/G¹⁹²的基因片段经酶切和测序发现，所构建的表达载体pLTK⁶³/G¹⁹² 阅读框架正确，且相应位点氨基酸获得了替换；经SDS-PAGE电泳检测，野生型LT及双突变体均表达出约33.0 ku和13.0 ku的两条蛋白带，与LTA、LTB亚基分子质量相吻合；经Western bolt检测，两个蛋白亚基均可与His抗体发生特异性反应；双突变蛋白的酶活性和毒性与野生型LT相比酶活性和毒性都有所降低；突变体LTK⁶³/G¹⁹²能辅助新城疫疫苗在血清和黏膜中产生较高抗新城疫病毒的IgG和IgA。说明LTK⁶³/G¹⁹²是一个良好的免疫佐剂。

Expression and Bio-activity of Escherichia coli Heat-labile Enterotoxin Mutant Proteins

In order to loose or nontoxic activity of E.coli heat-labile enterotoxin(LT) and remain its strong immunogenicity, mutants of E.coli heat-labile enterotoxin LTK⁶³/ G¹⁹² recombiant plasmids were made by gene SOE PCR or PCR. The target protein was expressed by IPTG inducing. The purified protein was checked in Western-blot detection, and ADP-ribosyltransferase activity and the toxicity assay of Patent-mouse test checked toxicity and enzyme activity. The ability of LTK⁶³/G¹⁹² LT as a mucosal adjuvant was tested by immunizing chickens intranasally using Newcastle vaccine for chickens as a bystander antigen. The results showed the positive recombinant plasmids LTK⁶³/G¹⁹² were confirmed the replacement of AGA (192) with GGA by PCR, enzyme digestion and nucleotide sequencing, and the expressed proteins were identified by SDS-PAGE. The purified protein subunits LTA and LTB were 33.0 ku and 13.0 ku. The purified protein was reacted with anti-His-tag antibodies in Western-blot detection. LTK⁶³/G¹⁹² enzyme activity and toxicity compared to wild-type LT significantly reduced. LTK⁶³/G¹⁹² and newcastle vaccine developed higher levels of serum and local antibodies to newcastle virus than NDV alone, which indicated LTK⁶³/G¹⁹² could be a good immunoadjuvant.