摘要
【目的】将透明颤菌血红蛋白(VHb)基因(vgb)在枯草芽孢杆菌中进行整合表达,提高β-半乳糖苷酶的产量。【方法】用枯草芽孢杆菌整合载体pA01和启动子P43构建vgb基因的整合表达载体pA-vgb,通过双交叉整合方式,将vgb基因整合到枯草芽孢杆菌DB104:bga的染色体上,构建枯草芽孢杆菌DB104:vbga。采用PCR和Southern blot对DB104:vbga进行检测,并通过发酵摇瓶试验研究VHb对β-半乳糖苷酶产量的作用。【结果】PCR与Southern blot检测结果表明,枯草芽孢杆菌DB104:vbga中vgb基因整合位置正确,且表达的VHb蛋白具有生物学活性。摇瓶试验结果表明,在转速250 r/min条件下,枯草芽孢杆菌DB104:vbga与DB104:bga的β-半乳糖苷酶活性无显著差异;在150 r/min的限氧条件下,vgb基因的表达促使DB104:vbga的β-半乳糖苷酶活性较DB104:bga提高了14.9%。【结论】vgb基因可用于提高枯草芽孢杆菌目的蛋白的产量。
[Objective] The study obtained Vitroscilla Hemoglobin gene integration expression in Bacillus subtilis for enhancing function in β-Galactosidase production. [Method] Using promoter P43 and pA01 ,an integrated vector of B. subtilis, pA-vgb, which was an integrated expression vector of vgb gene, was constructed. Using the method of double homologous recombination between plasmid pA-vgb and B. subtilis DB104 ：bga chromosome,vgb gene was integrated into DB104：bga,and then got a vgb-integrated expression strain named B. subtilis DB104：vbga. The result was identified by PCR analysis and Southern blot. Shake flask was used to study the function of VHb protein in production of β-Galactoside. [Result] B. subtilis DBl04：vbga was identified by PCR analysis and Southern blot,and the VHb protein had bio-activity. The results of shake flask showed that： at 250 r/rain aeration, the productions of β-Galactoside of B. subtilis DB104：vbga and DB104：bga were not significanty different;at 150 r/min aeration, the expression of vgb gene can improve β-Galactoside production,and the activity of β-Galactoside was 14.9% higher than that of the control. [Conclusion] In this study,it was clearly demonstrated that VHb significantly enhanced β-Galactosidase production by recombinant B. subtilis.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2009年第9期199-203,共5页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家重点新产品计划项目(2003ED760039)
