长双歧杆菌NCC2705株果糖结合蛋白BL0033基因的克隆及其在大肠杆菌中的表达

Gene Cloning and Prokaryotic Expression of the Fructose Binding Protein BL0033 from Bifidobacterium longum NCC2705

目的:克隆长双歧杆菌NCC2705株果糖结合蛋白BL0033的基因,利用大肠杆菌表达GST-BL0033融合蛋白并纯化。方法:以长双歧杆菌NCC2705株基因组为模板,PCR扩增BL0033基因,并将其插入pGEX-4T-1表达载体,转化至大肠杆菌DH5α;提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21,并对表达条件进行摸索;用谷胱甘肽-Sepharose4B树脂对可溶性GST-BL0033融合蛋白进行纯化。结果:PCR扩增的BL0033基因长度接近1000bp,与预期值一致;重组菌在IPTG浓度为0.05mmoL/L的条件下,于16℃诱导过夜后,SDS-PAGE分析可见可溶性表达条带,相对分子质量约60×103,与预期值一致;亲和纯化后,SDS-PAGE结果显示单一的表达条带。结论:克隆了BL0033蛋白的基因,并表达纯化了融合蛋白GST-BL0033,为进一步研究长双歧杆菌NCC2705株BL0033蛋白功能奠定了基础。

Objective： To clone the gene of fructose binding protein BL0033 from Bifidobacterium longum NCC2705, and express and purify fusion protein GST-BL0033 in E.coli BL21. Methods： The full-length coding sequence of BL0033 was amplified from the genome DNA of B.longum NCC2705 by PCR, and was cloned into the expression vector pGEX-4T-1. After sequencing, the positive recombinant plasmid was transformed into E.coli BL21 and was induced to express the fu- sion protein. The expression condition was optimized. Then, the fusion protein GST-BL0033 was analyzed by SDS-PAGE and further purified by GST-sepharose 4B beads. Results： The PCR product was about 1000 bp which was expected. The optimized inducing condition was under 0.05 mmol/L IPTG at 16℃for overnight. SDS-PAGE analysis revealed a specific and soluble expressing protein band with the molecular weight about 60 kD. The high purity GST-BL0033 was got through affinity beads. Conclusion： The recombinant protein GST-BL0033 was expressed in E.coli and purified. It laid foundation for further study on BL0033 of Bifidobacterium longum NCC2705.