大肠杆菌K88ac-ST_1-LT_B三价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 L TB基因 ,通过分离、纯化、内切酶酶切、连接和转化 ,构建了含 K88ac- ST1 - L TB融合基因表达载体的重组菌株 BL 2 1 (DE3) (p XKST3L T5 )。经酶切鉴定和 DNA序列分析证实 ,构建的重组质粒 p XKST3L T5中含有 K88ac- ST1 - L TB融合基因 ,且基因序列和阅读框架均正确。免疫试验结果表明 ,K88ac- ST1 - L TB融合蛋白能够诱发机体产生抗体 ,该抗体具有中和天然 ST1 肠毒素毒性的作用。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2 ML D的大肠杆菌强毒株 C8390 2 (K88ac,ST+ ,L T+ )的攻击。由此表明 ,构建的工程菌株 BL 2 1 (DE3) (p XKST3L T5 )

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 recognize ST 1 in vitro.These sera antibodies were able to neutralize the biological activity of native ST 1 in the suckling mouse assay.Mice immunized with crude inclusion bodies containing K88ac ST 1 LT B fusion protein or inactivated recombinant strain produced antibodies that were able to provide protection in a mouse model against the challenge of at least 2 MLD dose of Escherichia coli C83902.Hence the recombinant strain BL21(DE3)(pXKST3LT5) can be used candidate of vaccine strain.