枯草芽孢杆菌S44菌株ituD基因的克隆及原核表达

Cloning of ituD from Bacillus subtilis S44 and Its Expression in E.coil

为了进一步明确枯草芽孢杆菌S44菌株的防病机理,克隆其Iturin A合成关键基因ituD,对该基因进行了原核表达。通过PCR技术克隆了枯草芽孢杆菌S44菌株ituD基因,序列分析结果表明,该序列全长为1203bp,编码400个氨基酸,与已报道的ituD氨基酸序列同源性为85%。构建了原核生物表达载体pET28a(+)-ituD,对不同的IPTG诱导浓度和诱导时间等条件的优化结果表明,37℃6h能诱导ituD融合蛋白的最大量表达,在0.5mmol/LIPTG浓度下可以成功表达出ituD蛋白。

In order to explicit the mechanism of disease control of Bacillus subtilis S44,the ituD gene was cloned by PCR using specific primers and expressed in E. coil. The result showed that full-length of this gene sequence was 1203 bp,coded 400 amino acids,and had 85~ homology with reported sequence of ituD in GenBank. The ituD gene was inserted expression plasmid pET28a（＋） by restriction endonuclease digestion, electrophoresis, gel reclamation DNA ligation and so on, and then trans- formed it to t？.. coil BL21 （DE3）. The ituD fusion protein was expressed and the expression conditions were optimized. The re- sults showed that the expression level of ituD fusion protein reached the highest when cultured for 6 hours at 37 ~C, and 0.5 mmol/L IPTG could induce the expression of ituD in vitro.