大肠杆菌耐热性肠毒素STI三重串联突变体与黏附素K99融合基因的表达研究

Expression of Fusion Gene of Tree Heat-stable Toxin I’Mutants and K99 Adhesin of Enterotoxigenic Escherichia coli

产肠毒素大肠杆菌(ETEC)是一种导致仔畜和婴儿腹泻的主要病原之一,它的毒力因子主要有两类:黏附素(CFAs)和耐热性肠毒素(ST)或不耐热性肠毒素(LT)。通过PCR技术及双酶切连接技术,成功构建了含有3个STI突变体和1个黏附素K99基因的重组表达质粒pE3S(S)LK和pE3S(G)LK。重组菌株BL21(DE3)(pE3S(S)LK)和BL21(DE3)(pE3S(G)LK)的表达产物经SDS-PAGE和免疫印迹分析,表明以上两种重组菌株均能高效表达3STI(S)-K99和3STI(G)-K99融合蛋白,且融合蛋白能够被产肠毒素性大肠杆菌强毒株C83922抗血清特异性识别。其次,利用乳鼠灌胃实验检测重组蛋白的生物学毒性,结果均为阴性(G/C值≤0.083),这表明该菌株已无STI生物学毒性。这些为研发预防大肠杆菌性腹泻的新型高效多价基因工程疫苗提供了基本素材和理论指导。

Enterotoxigenic Escherichia coli（ETEC） is a major cause of diarrhea in animals and infants.The two most important virulence factors of ETEC are colonization factor antigens （CFAs）,and the heat stable （ST） enterotoxin or heat labile （LT） enterotoxin.The recombinant plasmid pE3S（S）LK and pE3S（G）LK,containing three STI’s mutants and one colonization factor K99 gene were gotten successfully by PCR and double enzyme cleaving and inserting technology.The result of SDS-PAGE and Western blotting proved that the recombinant strain BL21（DE3）（pE3S（S）LK） and BL21（DE3）（pE3S（G）LK） could produce the fusion protein of 3STI（S）-K99 and 3STI（G）-K99 effectively which were specially recognized by anti-serum of enterotoxigenic Escherichia coli C83922.In addition,the recombinant protein of 3STΙ（S）-K99 and 3STΙ（G）-K99 tuned out to lose their biological toxicity of STΙ（The numerical value of G/C≤0.083） by the test of pouring stomach of sucking mice.It provides basic materials and theoretical introduction for prevent colibacillus diarrhea of newborn piglet and contributes to developing a new effective multivalent gene engneering vaccine.