表达大肠杆菌K88ac-ST_1-LT_B融合蛋白基因工程菌株的构建

Construction of Recombinant Strain Expressing Enterotoxigenic Escherichia coli K88ac-ST_1-LT_B Fusion Protein

利用PCR技术 ,从大肠杆菌C8390 2质粒中扩增出K88ac基因、ST1 突变基因和LTB 基因 ,通过分离、纯化、内切酶酶切、连接和转化 ,构建了含K88ac ST1 LTB 融合基因表达载体的重组菌株BL2 1(DE3) (pXKST3LT5 )。经酶切鉴定和DNA序列分析证实 ,构建的重组质粒pXKST3LT5中含有K88ac ST1 LTB 融合基因 ,且基因序列和阅读框架均正确。经ELISA检测 ,重组菌株表达的K88ac ST1 LTB 融合蛋白能够被ST1 单抗、LTB 和K88ac抗体识别。经乳鼠灌胃试验证实 ,表达的融合蛋白已丧失天然ST1 肠毒素的活性。免疫实验结果表明 ,K88ac ST1 LTB 融合蛋白能够诱发小白鼠产生抗体 ,该抗体具有中和天然ST1 肠毒素的毒性作用 ,表明构建的重组菌株可以作为预防仔猪黄。

K88ac genes, heat stable enterotoxin I (ST 1) mutant genes and heat labile enterotoxin B subunit (LT B) genes from plasmids of Escherichia coli C83902 were amplified by PCR. The recombinant expression plasmid pXKST3LT5 containing K88ac ST 1 LT B fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3). The K88ac ST 1 LT B fusion protein was highly expressed in recombinant strain BL21(DE3)(pXKST3LT5) and the expression level of the K88ac ST 1 LT B fusion protein was about 75.53% of total cellular protein by SDS PAGE and thin layer gel scanning analysis. More importantly, mice immunized with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to re cognize ST 1 \%in vitro.\% These sera antibodies were able to neutralize the biological activity of native ST 1 in the suckling mouse assay. Hence the fusion protein was nontoxic and immunogenic, the constructed recombinant strain could be used as a candidate of vaccine strain.