流产布鲁氏菌BCSP31基因在大肠杆菌中的可溶性高效表达

High-Dissolved Expression of BCSP31 Gene of Brucella abortus in E. coli

设计1对引物,突变除去布鲁氏菌BCSP31(细胞表面蛋白31)基因的N端信号肽氨基酸序列,经NotⅠ和EcoRⅠ酶切后与相同处理的表达载体pGEX-4T-1连接,转化BL21(DE3)感受态细胞。酶切及PCR扩增鉴定,获得阳性克隆,测序证明其阅读框完全正确。用IPTG诱导表达构建成功的阳性克隆,对产物进行SDS-PAGE和Western-blotting检测,发现表达产物出现预期分子量57 kD的融合蛋白,该蛋白能与牛流产布鲁氏菌阳性血清发生反应,进一步证明目的蛋白主要以可溶性形式存在,表达量约占总蛋白的40%,应用GST琼脂糖凝胶FF纯化可得到纯度较高的表达产物。

The BCSP31 （brucella cell surfacial protein 31 ） of Brucella abortus were amplified by polymerase chain reaction. The amplified fragments were 906 bp in length. The fragments and expression vector pGEX-4T-1 were digested by the same restriction endonucleases. These genes were ligated and transformed into Escherichia coli. The insert position, the size and the reading frame were all corrected by PCR, restriction digestion and the sequence analysis. The result showed that the prokaryotic expression vectors were constructed successfully. Then the recombinants were transformed into BL21 （DE3） for BCSP31 expression with IFIG inducing. The expressed proteins were measured by SDS - PAGE and westernblotting. The results showed that the genes can express successfully in E. coli. The western-blotting results indicated that the expressed protein was recognized by the Brucella abortus positive serum. The rate of the expressed proteins in the induced bacteria protein was about 40%. The target products was resoluble fusion protein, and purified by the GST agarose gel FF.