Marc145细胞SHP-1、SHP-2结构域的克隆表达及抗体的制备与纯化  

Cloning and expression of the structure domain of SHP-1,SHP-2 from Marc145 cell and preparation of polyclonal antibodies

作　　者：杜东颖 崔春晓[1] 冯亚琼[1] 王冬梅[1] 郭嘉[1] 张莹莹[1] 夏平安[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2015年第10期1605-1609,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31172346)

摘　　要：应用RT-PCR技术从Marc145细胞中克隆出SHP-1、SHP-2结构域基因的cDNA序列,并将其亚克隆到原核表达载体pET-32a中,构建重组表达质粒pET-SHP-1、pET-SHP-2,转化大肠杆菌BL21,在IPTG的诱导下成功表达相对分子质量大小约为37 500、40 100的融合蛋白,将纯化过的融合蛋白分别免疫小鼠,制备鼠源血清,采用间接ELISA和Western Blot试验方法检测,ELISA测定多克隆抗体的血清效价为1∶12 800;Western Blot试验结果证明,制备的鼠抗pET-SHP-1、pET-SHP-2多克隆抗体可以与重组蛋白进行特异性结合,从而证明重组蛋白具有较好的免疫原性。采用硫酸铵盐析与DE-52离子交换层析相结合的方法提取并纯化血清得到纯度较高的鼠抗SHP-1、SHP-2抗体IgG。结果验证了Marc145细胞SHP-1、SHP-2的存在,克隆出了Marc145细胞SHP-1、SHP-2结构域基因并成功表达,为后续试验和研究奠定了基础。To verify the presence of SHP-1and SHP-2from Marc145 cells,cDNA sequences of SHP-1domain and SHP-2domain from the Marc145 cells were cloned by RT-PCR.The sequences were then subcloned into the prokaryotic expression vector pET-32 ato construct recombinant expression plasmid pET-SHP-1and pET-SHP-2and expressed in BL21.After induction with IPTG,we obtained the fusion proteins,which molecular mass were about 37 500 and 40 100,respectively.The polyclonal antibodies were obtained by immunizing mouse with the two purified fusion proteins and analyzed by indirect ELISA and Western blot.The results of ELISA showed that the antibody titers were both 1∶12 800.Western blot results indicated that the prepared polyclonal antibodies could bind to the recombinant proteins specifically,suggesting the recombinant proteins had good immunogenicity.The two-step method of ammonium sulfate precipitation method and the DE-52 ion exchange chromatography technology were used to extract and purify two specific antibodies.The results verified the presence of SHP-1and SHP-2from Marc145 cells,and the structure domains of SHP-1,SHP-2were cloned and expressed.

关 键 词：SHP-1 SHP-2 克隆 原核表达 多克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学]