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

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。

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

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.