摘要
目的:构建人转化生长因子β1(transforming growth factorβ1,TGF-β1)的原核表达载体,表达、纯化并鉴定该蛋白。方法:应用基因重组技术,构建人TGF-β1原核表达载体pQE30-TGF-β1,用SDS-PAGE电泳分析所表达蛋白,Ni-NTA亲和层析纯化该蛋白后用Western blot检测,同时用该蛋白免疫BALB/c小鼠,测定免疫血清效价。结果:成功构建了人TGF-β1的原核表达载体pQE30-TGF-β1,表达的蛋白经SDS-PAGE电泳分析,在约Mr14kDa处出现了一条新生的蛋白条带。经Ni-NTA亲和层析纯化后,Western blot检测显示,重组蛋白6×his-TGF-β1能很好地与抗TGF-β1单克隆抗体特异性结合,并能够诱导BALB/c小鼠产生抗人TGF-β1的中和抗体,免疫血清的效价为1:10000。结论:转化生长因子β1是一种多功能细胞因子,在肿瘤的免疫逃避中发挥重要作用。
Objective：To construct prokaryotic expression vector pQE30 -TGF - β1 of transforming growth factor β1 （ TGF- β1 ）, expression, purification were tested. Methods： To construct prokaryotic expression vector pQE30 -TGF- β1 and express in E. coli, purify fusion protein 6 × his -TGF- β1, detect it with anti -hTGF- β1 monoclonal antibody and immune BALB/c mice with it. Results. We successfully constructed prokaryotic expression vector pQE30 - TGF - β1 and expressed in E. coil Purified fusion protein 6 × his - TGF - β1 had a good reactivity with anti - hTGF - β1 monoclonal antibody and can induce the production of antibody in the body of BALB/c mice. The titer of immune sera was 1：10000. Conclusion： Transforming growth factor β1 （ TGF -β1 ）is a potent immunosuppressive cytokine that has a pivotal role in tumorigenesis , development and metastasis. The successful cloning and expression of rhTGF - β1 gene and purification of 6 × his - TGF - β1 protein lay a basis for the research of autovaccine of TGF - β1.
出处
《现代肿瘤医学》
CAS
2008年第9期1460-1462,共3页
Journal of Modern Oncology
