表达大肠杆菌肠毒素ST_1—LT_B融合蛋白工程菌株的免疫原性研究

Studies on the Immunogenicity of the Genetic Engineering Strain Expressing Escherichia Coli Heat Stable Enterotoxin Ⅰ and Heat Labile Enterotoxin B subunit Fusion Protein

已构建的能表达大肠杆菌肠毒素ＳＴ１—ＬＴＢ融合蛋白的工程菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴ－ＳＬＴ１）及其表达产物经动物试验证实没有毒性反应。用从ＩＰＴＧ诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原，免疫小鼠，结果免疫小鼠至少能抵抗１．５ＬＤ１００的大肠杆菌强毒株Ｃ８３９０２（Ｋ８８ａｃ，ＳＴ＋，ＬＴ＋）的攻击。用提取的包涵体免疫家兔后，采集的血清能够中和天然ＳＴ１的毒性。这表明构建的工程菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴＳＬＴ１）可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株。

おhe authors used restriction endonucleases EcoR Ⅰ and Hind Ⅲ to cleave 084kb E.coli ST 1—LT B fusion gene from plasmid pXSLT1,isolated by 15% agarose gel electrophoresis,recovered 084kb gene fragment,then inserted it into an expression vector pET—28b(+) which cleaved with EcoR Ⅰ and Hind Ⅲ by blunt—end ligation.The recombinant plasmid pXET—SLT1 was studied in detail by restriction endonuclease analysis.The results showed that the recombinant plasmid carried ST1—LTB fusion gene.By transformation of E.coli BL21(DE3),we obtained E.coli BL21(DE3)(pXETSLT1).The recombinant strain can produce pre-ST1—LTB fusion protein by ELISA and SDS-PAGE,and the fusion protein was nontoxic.After recombinat strain was induced by IPTG,is expressed product was about 3321% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.