【摘要】 目的:以金黄色葡萄球菌基因组DNA为基础,在枯草芽孢杆菌中得到具有剪切功能的金黄色葡萄球菌丝氨酸类蛋白酶B。方法:以金黄色葡萄球菌基因组DNA为模板,PCR扩增得到SplB基因,同源重组到表达载体pHTSHIS上,再转化枯草芽孢杆菌;优化诱导条件发酵枯草芽孢杆菌,离心后超滤浓缩上清液,再经镍柱分离纯化;利用SDS-PAGE、Lowry法对蛋白的相对分子质量、浓度等进行分析,对蛋白的酶切活性进行鉴定。结果:金黄色葡萄球菌基因组DNA经PCR扩增得到与预计大小相符的DNA片段,通过同源重组构建了表达载体pHTS-SplB-His;选取诱导前菌液D_600nm为2.0、IPTG浓度为0.5 mmol/L、诱导4 h的最优条件发酵枯草芽孢杆菌,表达大量金黄色葡萄球菌丝氨酸类蛋白酶B;蛋白浓缩后,经镍柱分离纯化得到纯度较高且具有剪切功能的金黄色葡萄球菌丝氨酸类蛋白酶B。结论:采用枯草芽孢杆菌进行发酵表达,可提高金黄色葡萄球菌丝氨酸类蛋白酶B的表达量,并且能够得到具有剪切功能的蛋白。本研究可为外源蛋白在枯草芽孢杆菌中的发酵技术提供重要参考。更多还原

【Abstract】 Objective: To obtain serine protease-like B（SplB） with shear function in Bacillus subtilis based on the genomic DNA of Staphylococcus aureus（Sa）. Methods: The SplB gene was obtained by PCR amplification using S.aureus genome DNA as template and subcloned into the expression plasmid pHTS-His vector, then the plasmid was transformed into B.subtilis. The conditions were optimized for the fermentation of B.subtilis. The supernatant was concentrated by ultrafiltration after centrifugation and purified by nickel column. The molecular weight was analyzed by SDS-PAGE. The concentration was determined by Lowry. The activity of the protein was identified by enzyme digestion. Results: The genomic DNA of S.aureus was amplified by PCR to obtain a DNA fragment corresponding to the expected size. The expression vector pHTS-SplB-His was constructed by homologous recombination. Optimally, induction was iniatiated at D_600nm of 2.0 by the final IPTG concentration of 0.5 mmol/L for 4 h. After the expression product was concentrated, separated and purified by nickel column, the Sa SplB with high purity and shear function was obtained. After the protein was concentrated, it was separated and purified by nickel column to obtain Sa SplB with high purity and shear function. Conclusion: The Sa SplB can be enhanced in the expression host B.subtilis in addtion, the protein has cleavage function. This study may provide an important reference for the fermentation of exogenous protein in B.subtilis.更多还原