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目的构建能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因的载体,并用此载体在大肠埃希菌和双歧杆菌中表达人白介素-10基因(hIL-10)的蛋白产物;为hIL-10基因重组双歧杆菌治疗炎症性肠病做前期准备。方法以质粒pDG7为模板扩增pMB1片段,构建表达质粒pET28B1。用PCR法扩增hIL-10基因,将此目的基因以及pET28B1经酶切后用连接酶连接,形成重组质粒pET28B1-hIL10。pET28B1-IL10转染大肠埃希菌BL21和长双歧杆菌。最后用Western blot检测hIL-10基因在大肠埃希菌和长双歧杆菌中的表达情况。结果pET28B1-hIL10阳性克隆扩增后提取质粒并进行基因测序,结果显示插入片段为hIL-10,序列正确且无突变。hIL-10基因在大肠埃希菌、长双歧杆菌中的诱导表达产物通过Western blot检测验证为IL-10蛋白,显示该hIL-10表达载体在大肠埃希菌阳性克隆中经诱导可高量表达,在长双歧杆菌体中有少量表达。结论成功构建质粒pET28B1,该质粒能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因hIL-10。
OBJECTIVE: To construct a vector capable of shuttling the target gene in Escherichia coli and Bifidobacterium and express the protein product of human interleukin-10 gene (hIL-10) in Escherichia coli and Bifidobacterium using this vector; -10 gene recombinant bifidobacteria treatment of inflammatory bowel disease to do the preliminary preparation. Methods The pMB1 fragment was amplified by using the plasmid pDG7 as a template to construct the expression plasmid pET28B1. The hIL-10 gene was amplified by PCR, and the target gene and pET28B1 were ligated and ligated to form the recombinant plasmid pET28B1-hIL10. pET28B1-IL10 was transfected into Escherichia coli BL21 and Bifidobacterium longum. Finally, the expression of hIL-10 gene in Escherichia coli and Bifidobacterium longum was detected by Western blot. Results The plasmid pET28B1-hIL10 was amplified and sequenced. The result showed that the inserted fragment was hIL-10 with correct sequence and no mutation. The induced product of hIL-10 gene in Escherichia coli and Bifidobacterium longum was confirmed to be IL-10 protein by Western blot, indicating that the hIL-10 expression vector can be induced in Escherichia coli positive clones in high amounts The expression of Bifidobacterium longum in a small amount of expression. Conclusion The plasmid pET28B1 was successfully constructed and successfully transfected into Escherichia coli and Bifidobacterium to express hIL-10 gene.