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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 one of the major causative agents of diarrhea in young animals and infants. There are two major virulence factors: adhesins (CFAs) and heat-tolerant enterotoxins (ST) or impatience Heat enterotoxin (LT). The recombinant expression plasmids pE3S (S) LK and pE3S (G) LK containing three STI mutants and one adhesin K99 gene were successfully constructed by PCR and double enzyme digestion. The expressed products of recombinant strains BL21 (DE3) (pE3S (S) LK) and BL21 (DE3) (pE3S (G) LK) were analyzed by SDS-PAGE and Western blotting. ) -K99 and 3STI (G) -K99 fusion proteins, and the fusion protein can be specifically recognized by the antiserum to enterotoxigenic E. coli virulent strain C83922. Second, the biological toxicity of the recombinant protein was tested by intragastric administration in the suckling mice, and the result was negative (G / C value≤0.083), indicating that the strain had no biological toxicity to STI. These provide the basic material and theoretical guidance for the development of a novel and highly efficient multivalent genetic engineering vaccine for preventing colibacillosis.