摘要
目的克隆小鼠mlL-35两亚基mEBI3，mlL-12p35cDNA，并构建mlL-35基因的原核表达载体并进行诱导表达。方法RT—PCR方法从RAW246．7细胞中扩增mEBI3基因及脂多糖体外刺激BALB／c小鼠的脾细胞中扩增mlL-12p35基因，通过重叠PCR方法扩增出mEBI3-Linker-mlL-12p35基因，并构建原核表达载体pET-30a．mlL-35，转化感受态E．coliBL21（DE3），并用IPTG进行诱导表达。SDS-聚丙烯酰胺凝胶电泳方法检测目的蛋白的表达。结果成功克隆了mEBI3、mlL-12p35cDNA，通过重叠PCR方法扩增出mEBI3-Linker—mlL-12p35片段，并构建出原核表达载体pET-30a-mlL-35；在IPTG诱导下在E．coliBL21（DE3）中高效表达相对分子质量（Mr）为50000的重组mlL-35蛋白，目的蛋白主要以包涵体的形式存在于沉淀中。结论利用构建的原核表达载体pET-30a-mlL-35成功表达出mlL-35蛋白，为进一步探讨mlL-35的生物学功能奠定了基础：
Objective To construct prokaryotic expression vector of IL-35 gene and to express protein IL-35 with prokaryotie expression system. Methods mEBI3 gene was amplified from RAW246.7 cells and IL-12p35 gene was from spleen cells of BALB/c mouse stimulated by LPS by RT-PCR. pcDNA3. I/V5-HisB-mEBI3 and pcDNA3, 1/V5-HisB-mlL-12p35 were constructed. Then mEBI3-LinkermlL-12p35 gene was amplified by overlapping PCR,and pET-30a-mlL-35 prokaryotic expression vector was constructed. The vector was transformed into E. coli BL21 （ DE3 ） , induced by IPTG to express protein. SDS-polyaclylamide gel electrophoresis was used to observe the expres- sion of mlL-35 protein. Results The prokaryotic expression vector pET-30a-mlL-35 was successfully constructed ; the induced and expressed protein matched well with the predicted relative molecular mass of mouse IL-35 in E. eoli BL21 （ DE3 ） by the IPTG. The protein mainly existed in the precipitate in the form of inclusion body. Conclusion The vector of rolL-35 gene was constructed successfully and the protein was successfully expressed in prokaryotic expression system.
出处
《潍坊医学院学报》
2010年第5期324-327,共4页
Acta Academiae Medicinae Weifang
基金
潍坊医学院研究生创新基金（YC2008014）
关键词
mIL-35
载体构建
原核表达
mlL-35
Vector construction
Prokaryotic expression
