β-半乳糖苷酶在枯草芽孢杆菌中的分泌表达

Expression and Secretion of β-galactosidase in Bacillus subtilis

为检测本课题组构建的枯草芽孢杆菌分泌表达载体pGPST中启动子和信号肽的活性,将β-半乳糖苷酶基因插入表达载体的多克隆位点,构建含有β-半乳糖苷酶基因的重组质粒pGPST-lacZ,重组质粒转化枯草芽孢杆菌,分别测定液体培养基中上清液和菌体沉淀中β-半乳糖苷酶的活性。培养上清液中的酶活在22h达到峰值,约为26mU/ml,之后开始下降;菌体沉淀中的酶活在14h最高,达6mU/ml,而阴性对照pGPST没有检测到酶活性。结果表明分泌型表达载体中的启动子和信号肽具有较好的活性,能实现异源基因在枯草芽孢杆菌中的分泌表达。本试验为该表达载体的进一步研究和应用提供重要的数据资料,也为外源基因在枯草芽孢杆菌中的表达提供参考资料。该载体将在研究外源基因在枯草芽孢杆菌中表达发挥重要作用。

To characterize the promoter and signal was inserted into vector pGPST yielding the recombinant -peptide activity of expression vector pGPST, lacZ gene plasmid pGPST-lacZ and it was translated into Bacillus subtilis AS 1700 by electroporation. The active of ^-galactosidase in culture supernatant and bacteria were analyzed respectively. In culture supernatant the enzyme active reaches peak after 22h, approximately 26 mU/ml. then decrease slowly. In bacteria pellet the most expression was almost 6mU/ml after cultured 14h. While none active was detected in negative control. The results show that the promoter and signal-peptide can be used to express heterologous gene in Bacillus subtilis. The study provides considerable data which are useful for the further development of the expression vector pGPST and the study of foreign gene expression in Bacillus subtilis. The vector pGPST will play a significant role in gene expression in Bacillus subtilis.