大肠埃希菌—双歧杆菌穿梭表达载体的构建及白介素-10基因的表达

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。

Construction of a novel expression vector which can produce human IL-10 in both Escherichia coli and Bifidobacterium longum

Objective To construct a novel vector which could express human IL-10 gene in both Escherichia coli and Bifidobacterium longum, prepare for the treatment of inflammatory bowel disease with bifidobacterium transferred by interleukin-10 gene. Method The pMB1 fragment was amplified by PCR with plasmid pDG7 as a template to construct a novel expression vector (pET28B1). After amplifying with PCR, the human IL-10 gene and pET28B1 were excised by both EcoR I and BamH I restriction and ligated with each other to finish the construction of pET28B1-hIL10. pET28B1-hIL10 was transformed into both Escherichia coli and Bifidobacterium longum. Protein fractions were separated by SDS-PAGE and electroblotted onto nitrocellulose membrane for Western blot analysis. Result The inserted element was hIL-10 gene with the correct sequence and no mutation. The protein fraction which was expressed in both Escherichia coli and Bifidobacterium longum was hIL-10 protein by Western blot. The hIL-10 protein expression was more sufficient in Escherichia coli than in Bifidobacterium longum. Conclusion A novel vector pET28B1 was constructed successfully, which can express human IL-10 gene in both Escherichia coli and Bifidobacterium longum.