K88菌毛蛋白亚基基因克隆、表达及重组蛋白抗血清制备

CLONING , EXPRESSION OF K88 FIMBRIAL SUBUNIT GENE AND PREPARATION OF THE ANTISERUM OF THE RECOMBINANT PROTEIN

利用 PCR技术 ,从仔猪黄痢的致病菌中扩增出不含信号肽序列的 K88菌毛蛋白亚基基因片段 ,将其克隆到 E.coli表达载体 p ET2 8a(+)中 ,构建了该基因的原核表达载体 p882 8,导入 E.coli BL 2 1(DE3) ,得到工程菌株。 SDS- PAGE分析结果显示 ,经 IPTG诱导 ,该亚基基因高效表达出重组 K88菌毛蛋白 ,约占菌体总蛋白的 30 %。用含重组蛋白的凝胶作为抗原 ,免疫新西兰大白兔 ,首次制备 K88菌毛蛋白亚基的抗血清。免疫学分析表明 。

The DNA fragment of K88 fimbrial subunit gene without signal peptide sequence was amplified by PCR from the DNA of enterotoxigenic E.coli K88ac strain. After DNA cloning and sequencing, the fragment was inserted into E.coli expression vector pET28a(+) . The recombinant expression plasmid p8828 was transferred into E.coli strain BL21(DE3), the over expressed recombinant protein was detected in the soluble protein of the expression strain induced by IPTG, then the protein purified using preparation electrophoresis. The antiserum against the recombinant protein was raised in a rabbit. With the immunological analysis, the antiserum showed specific immunological response to standard enterotoxigenic E.coli strain. This work provides a new method for preparing genetic engineering vaccine to prevent diarrheal disease in piglets.