影响大肠杆菌不耐热肠毒素2型毒性的氨基酸初步筛选

Preliminary screening of the key amino acids affecting heat labile enterotoxin type 2 toxicity of Escherichia coli

为探究影响产肠毒素大肠杆菌(ETEC)不耐热肠毒素2型(LT2)毒性的关键氨基酸及其突变重组蛋白的免疫原性,本研究扩增了LT2编码基因elt2,并采用重叠延伸PCR(SOE PCR)方法分别对其A亚基进行R5K、H42A、S59K、V95K、Y102K和E110K的单突变,将elt2及其6个突变基因分别双酶切后克隆于pET-30a中构建重组大肠杆菌pET-30a-elt2/BL21、pET-30a-R5K/BL21、pET-30a-H42A/BL21、pET-30a-S59K/BL21、pET-30a-V95K/BL21、pET-30a-Y102K/BL21、pET-30a-E110K/BL21,经IPTG诱导表达了重组LT2(rLT2)及其突变重组蛋白mrLT2-R5K、mrLT2-H42A、mrLT2-S59K、mrLT2-V95K、mrLT2-Y102K、mrLT2-E110K;利用细胞毒性试验和小鼠致病性试验比较了rLT2和突变重组蛋白对小鼠肾上腺皮质瘤(Y1)细胞的毒性作用及对小鼠的致病性,并将rLT2和mrLT2-V95K作为免疫原接种小鼠检测其对小鼠的免疫原性。结果显示,突变重组蛋白对Y1细胞的毒性作用均弱于rLT2,其中mrLT2-V95K对Y1细胞的毒性作用最弱为1.145×10^(1)TCID_(50)/0.1 mL;rLT2及该突变重组蛋白对小鼠均无致病性,表明小鼠也不适合用于LT2的致病性检测。mrLT2-V95K免疫BALB/c小鼠的血清特异性IgG抗体效价和对rLT2的中和抗体效价分别为1:102 400和1:32,与rLT2的免疫原性无显著差异。本研究首次证实LT2 A亚基的aa95对LT2细胞毒性作用的影响较明显,其突变重组蛋白具有良好的免疫原性,为大肠杆菌减毒疫苗研究提供了重要科学信息。

In order to explore key amino acids affecting the toxicity of heat labile enterotoxin type 2 (LT2) of enterotoxigenic Escherichia coli (ETEC) as well as the immunogenicity of the mutant recombinant toxins,LT2 coding gene elt2 was amplified,and its A subunits amino acid 5,42,59,95,102 and 110 were mutated by overlapping extension PCR (SOE PCR).After double enzyme digestion,elt2 and its mutant variants were inserted into pET-30a to construct recombinant plasmids pET-30a-elt2/BL21pET-30a-R5K/BL21,pET-30a-H42A/BL21,pET-30a-S59K/BL21,pET-30a-V95K/BL21,pET-30a-Y102K/BL21,pET-30a-E110K/BL21,respectively.Recombinant LT2 (rLT2) and its mutant recombinant proteins mrLT2-R5K,mrLT2-H42A,mrLT2-S59K,mrLT2-V95K,mrLT2-Y102K,mrLT2-E110K were induced by IPTG.The cytotoxicity and pathogenicity of rLT2 and mutant recombinant protein to mouse adrenocortical tumor (Y1) cells were compared by cytotoxicity test and mouse pathogenicity test,respectively.The rLT2 and mrLT2-V95K were inoculated into mice as immunogens to determine their immunogenicity to mice indicating that mice are not suitable for LT2 pathogenicity detection.The results showed that the toxic effects of mutant recombinant protein on Y1 cells were weaker than rLT2,and the toxic effects of mrLT2-V95K on Y1 cells were the weakest,which is 1.145×10^(1)TCID_(50)/0.1m L.r LT2and its mutant recombinant proteins were not pathogenic to mice.The serum specific Ig G antibody titers and neutralizing antibody titers against r LT2 in mr LT2-V95K immunized mice were 1:102 400 and 1:32,respectively,which had no significant difference from the immunogenicity of rLT2.This study confirmed for the first time that LT2A subunit aa95 had an obvious effect on LT2 cytotoxicity,and its mutant recombinant protein has good immunogenicity,which provides important scientific information for the research of attenuated ETEC vaccine.