大肠杆菌不耐热肠毒素突变体的表达及活性研究

Expression and bio-activity of Escherichia coli heat-labile enterotoxin mutants proteins

利用重叠延伸PCR扩增技术,体外获得大肠杆菌不耐热肠毒素(LT)突变体LTK63、LTR72和LTG192全基因片段,酶切和测序结果表明构建的表达载体pET30a-LT、pET30a-LTK63、pET30a-LTR72、pET30a-LTG192阅读框架正确,且相应位点氨基酸获得了替换。诱导表达产物用SDS-PAGE检测,野生型LT蛋白及3个突变体均表达出约33.0Ku和13.0Ku的2个蛋白带,与LTA、LTB亚基分子量大小相吻合;Westernblot检测,2个蛋白带均可与抗His抗体发生特异性免疫反应,而13.0Ku的蛋白带还可与抗霍乱毒素(CT)抗体发生免疫学反应。ADP-核糖转移酶活性试验分析,各突变体蛋白酶活性均低于野生型LT蛋白,但仍保留一些酶活性。Patent-mouse毒性试验检测,LTK63、LTR72和LTG192突变体蛋白的毒性均有不同程度降低,LTK63毒性降低最显著,LTG192蛋白毒性相对较大。

Mutants of Escherichia coli heat-labile enterotoxin （LT） LTK63, LTR72 and LTGI92 recombiant plasmids were constructed by gene SOEing PCR. The positive recombinant plasmid pET30a-LT, pET30a-LTK63, pET30a-LTR72 and pET30a- LTG192 were constructed, where TCT （63）, GCA （72） and AGA （192） were replaced with AAA, CGT and GGA respectively, The plasmids were transformed into the host E.coli. BL21 （DE3） respectively, and protein expression were identified by SDS-PAGE. The expressed protein subunits LTA and LTB were purified with anti-His-tag antibodies, The results showed that two protein subunits have the same immune activity as wild LT protein. ADP-ribosyltransferase activity test showed that LTK63, LTR72 and LTGI92 all have less bioactivity than that of wild LT. Test in Patent-mouse showed that the toxicity of LTK63, LTR72 and LTG192 were all significantly reduced, with LTK63＇ being the lowest among the three mutants proteins.