表达ST1-LTB-α-β融合蛋白基因工程菌株的构建及其免疫原性分析

Construction and Immunogenicity of a Genetic Engineered Strain Expressing Nontoxic ST_1-LT_B-α-β Fusion Protein Against Diarrhea of Piglet

采用分子生物学技术构建了含有ST1-LTB-α-β融合基因的重组菌株BL21(DE3)(pETST3LTBαβ),SDS-PAGE和Western blotting分析表明ST1-LTB-α-β融合基因在大肠杆菌中得到了高效表达,融合蛋白分子量约为110kD;20L发酵罐培养得到的最佳诱导条件为:重组菌株以1%接种量、5L/min通气量培养3h后加终浓度为0.03mol/L的乳糖诱导,通气量升至12.5L/min继续培养6h,表达量占菌体总蛋白的38.53%;表达的ST1-LTB-α-β融合蛋白无毒性但具有免疫原性,可以抵抗大肠杆菌和产气荚膜梭菌的感染;构建的重组菌株BL21(DE3)(pETST3LTBαβ)有望作为预防仔猪腹泻基因工程疫苗的候选生产菌株。

We constructed a recombinant strain BL21 （DE3） （pETST3LTBαβ） including ST1-LTB-α-β fusion gene via molecular technology. The SDS-PAGE and Western blotting indicated that the ST1-LTB-α-β fusion protein was highly expressed in Escherichia coli and the molecular weight of the fusion protein was about 110 kD. The recombinant strain was induced in different concentrations of lactose and different aeration rate. The optimal culture conditions in 20 L fermentor were 1% inoculation （V/V）, initial aeration 5 L/min, 0.03 mol/L lactose addition 3 hours after inoculation, and increased the aeration to 12.5 L/min for the following 6 hours. The fusion protein was about 38.53% of total cellular protein. It was nontoxic, immunogenic and protective against enterotoxigenic E. coli and Clostridium perfringens infection The constructed recombinant strain BL21 （DE3） （pETST3LTBαβ） could serve as a candidate vaccine strain against diarrhea of piglet.