大肠杆菌肠毒素ST_1、LT_B基因融合及其免疫原性研究

Gene Fusion of Heat-stable Enterotoxin Ⅰ Gene and Heat-iabile Enterotoxin B Subunit Gene from Enterotoxigenic Escherichia coli and Its Immunogenicity

目的:构建大肠杆菌肠毒素ST1-LTB融合基因,并研究其表达产物的免疫原性。方法:采用PCR和基因突变技术,从大肠杆菌C83902质粒中扩增ST1突变基因和LTB基因,通过基因分离、纯化、内切酶酶切、连接和转化,构建含ST1-LTB融合基因表达载体的重组菌株,并用酶切和DNA序列分析鉴定重组质粒,同时用ST1-LTB融合蛋白粗提物免疫小鼠,观察免疫攻毒保护效果。结果:构建了含ST1-LTB融合基因表达载体的重组菌株BL21(DE3)(pXSL1),ST1-LTB融合基因的序列和阅读框架均正确,其表达的ST1-LTB融合蛋白能够被ST1单抗和LTB抗体识别,且该融合蛋白已丧失天然ST1肠毒素的活性。ST1-LTB融合蛋白能够诱发小鼠产生抗体,该抗体具有中和天然ST1肠毒素的毒性作用。结论:构建的重组菌株BL21(DE3)(pXSL1)可以高效表达ST1-LTB融合蛋白,其表达产物ST1-LTB融合蛋白具有良好的免疫原性,为更有效地预防仔猪黄痢提供了一种新型基因工程菌苗候选菌株。

Objectives：To construct ST1 - LTB fusion gene of Escherichia coli enterotoxin and study its immunogenicity of the expressed products. Methods： To amplify heat- stable enterotoxin T （ST1） mutant gene and heat- labile enterotoxin B suburtit （LTB） gene with PCR. To construct the recombinant expression plasmid pXSL1 containing Sit - LTB fusion gene with recombinant DNA technique and identify with endonucleases digestion and sequencing. To study immunogenicity of ST1 - LTB fusion protein by mice immuniaed with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain.Results：The recombinant expression plasmid pXSL1 containing ST, - LTB fusion gene was constructed. The ST1 - LTB fusion protein was highly expressed in recombinant strain BL21 （DE3） （pXSL1） and the expression level of ST1 - LTB fusion protein was about 33.21% of total cellular protein by SDS- PAGE and thin- layer gel scanning analysis. Mice immuniaed with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to recognize ST1 in vitro. These sere antibodies were able to neutralize the biological activity of native sr, in the suckling mouse assay. Conclusion： The ST1 - LTB fusion protein was produced by recombinant strain BL21 （DE3） （ pXSL1 ） and was nontoxic and immunogenic. The constructed recombinant strain could be used as a candidate of vaccine strain.