Fas胞外区基因的构建、表达、纯化及多克隆抗体的制备  

Construction,expression,purification and polyclonal antibody preparation of Fas extracellular region gene

作　　者：张佳锴[1,2] 孟庆宇[1] 程小峰[1] 刘瑞振[1] 庄国洪[1] 

机构地区：[1]厦门大学医学院抗癌研究中心,福建厦门361005 [2]厦门大学生命科学学院,福建厦门361005

出　　处：《中国生化药物杂志》2010年第1期35-39,共5页Chinese Journal of Biochemical Pharmaceutics

基　　金：厦门市科技计划项目(3502Z20083008)

摘　　要：目的构建Fas胞外区(eFas)基因的表达载体,表达纯化重组蛋白,进行多克隆抗体的制备,为进一步功能研究奠定基础。方法通过重叠PCR获得eFas基因的编码序列,构建pET-22b(+)/eFas表达载体,转化大肠杆菌Rosetta-gami,IPTG诱导表达,Ni-NTA柱亲和纯化,SDS-PAGE鉴定重组蛋白的纯度。将纯化的eFas融合蛋白免疫新西兰白兔制备多克隆抗体,通过ELISA方法检测多克隆抗体的效价。结果获得了eFas的编码序列与表达载体,目的蛋白主要在包涵体中表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度达95%以上。结论eFas融合蛋白基因的构建、表达、纯化以及多克隆抗体的制备,为进一步研究Fas提供了材料。Purpose To construct expression vector of Fas extracellular region gene（eFas）, to express and purify recombination protein and to prepare polyclonal antibody, which have laid a foundation of studying its function. Methods The eFas gene encoding sequence was acquired through overlapping PCR, and pET-22b （ ＋ ）/eFas expression vector was constructed. Then this vector was transformed into E. coli Rosetta-gami. Recombinant protein was expression being induced by IPTG, and was purified using Ni-NTA matrix of affinity chromatograph. The purity of recombination protein was identified by SDS-PAGE. Hereafter,the purified eFas recombinant protein was immunized to New Zealand white rabbit in order to prepare polyclonal antibody. The titer of polyclonal antibody was determined by ELISA. Results The encoding sequence and expression vector of eFas was obtained while the interest protein was mainly expressed in the inclusion body. The eFas fusion pro- tein＇s expression quantity accounts for more than 30% proportion of total E. coli protein. The eFas protein we obtained was provided with the purity of at least 95 %. Conclusion The successful constrution, expression and purification of FasL fusion protein and preparation of polyclonal antibody will provide some material for further studies of Fas.

关 键 词：FAS 细胞凋亡 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]