| 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。 |
| 关键词: 透明颤菌 枯草芽孢杆菌 vgb基因 同源重组 |
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| 基金项目:国家重点新产品计划项目(2003ED760039) |
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| Integrated expression and roles of vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis |
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| Abstract: |
| 【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 DB104: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/min 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. |
| Key words: Vitroscilla B.subtilis vgb gene integration |
