BALB/C鼠冷诱导RNA结合蛋白的原核表达、纯化及其单克隆抗体的制备  

Prokaryotic Expression,Purification and Preparation of Monoclonal Antibodies of Cold Inducible RNA-binding Protein in BALB/C Mice

作　　者：李士泽[1] 赵巧香[1] 辛九庆[2] 赵雅楠[1] 尹位[1] 杨焕民[1] 计红[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]中国农业科学院哈尔滨兽医研究所,哈尔滨150000

出　　处：《中国生物工程杂志》2010年第3期33-39,共7页China Biotechnology

基　　金：大庆高新区创新基金(DQGX08YF038)资助项目

摘　　要：为了获得冷诱导RNA结合蛋白(CIRP)的单克隆抗体,通过RT-PCR方法从冷处理BALB/C小鼠睾丸的总RNA中扩增获得CIRP基因序列,克隆至pGEM-T载体,获得重组质粒pGEM-CIRP,并进行序列测定。将测序正确的CIRP序列插入质粒pQE-30-Xa,构建表达载体pQE-30-CIRP并转化E.coliM15,IPTG诱导后SDS-PAGE分析表明CIRP基因在大肠杆菌中获得高效表达,可溶性分析表明CIRP融合蛋白以包涵体形式表达。采用Ni-NTA亲和层析、电洗脱纯化融合蛋白CIRP,将纯化的蛋白免疫BALB/C小鼠,三次免疫后,间接ELISA测定小鼠效价,效价为1:105,采用杂交瘤技术融合,用间接ELISA法、有限稀释法筛选阳性杂交瘤细胞,通过Western blot对McAb特异性进行鉴定,利用间接ELISA方法对McAb的Ig亚类(型)、杂交瘤细胞上清、腹水效价进行测定。结果获得了1A6、2D3、3C5及4C44株稳定分泌CIRPMcAb的杂交瘤细胞株,1A6、3C5、4C4产生的单克隆抗体亚类均为IgG2b,2D3产生的单抗亚类为IgG3,轻链均属κ链。4株单克隆细胞上清效价均在1:103以上,腹水效价均达1:107以上。Westernblot分析4株单抗均能与重组蛋白CIRP特异性结合,与空载体对照没有反应。以3C5细胞株的腹水为一抗,检测冷应激小鼠多种组织中天然CIRP蛋白表达,结果显示3C5细胞腹水能识别心、肝、脾、肺、肾、脑、睾丸等多种组织中天然CIRP,并与能之与结合。这证明该抗体具有良好特异性和结合活性,制备的单克隆抗体可以用于深入开展CIRP的功能性和应用性研究。The CIRP gene of mouse was amplified from testis total RNA of BALB/C mouse by RT-PCR.PCR product was cloned into the pGEM-T vector to construct recombinant plasmid pGEM-CIRP for sequencing.The correct CIRP sequence was inserted into pQE-30-Xa to construct expression vector pQE-30-CIRP.It was transformed into host strain E.coli M15 and was induced by IPTG.It shows efficient expression in the E.coli by SDS-PAGE detected.The expression product exists in inclusion body.CIRP fusion protein was purified by Ni-NTA affinity chromatography and electroelution.The purified CIRP protein was used to immune BALB/C mice three times.Result of indirect ELISA shows that antibody titers of serum was 1∶105.The hybridoma technique was adopted to fusion.Positive hybridoma cell was screened by indirect ELISA and 3 times subcloning.Western blot was used to indentify McAb specificity.Indirect ELISA was used to identify McAb IgG subclasses and determine titer of McAb cell supernatants and ascites.1A6,2D3,3C5 and 4C4 4 hybridoma cell line of stable secretion CIRP McAb was obtained.The antibody subclass of 1A6,3C5,4C4 were IgG2b equally,The antibody subclass of 2D3 was IgG3,light chains were κ chain equally.4 monoclonal cell supernatant titers were more than 1∶10^3,ascites titer reaching more than 1∶107.Western blot analysis indicates four monoclonal antibodies can specifically combine with recombinant protein CIRP and had no response with void vector comparison.The ascites of cell line 3C5 was as primitive antibody to test natural CIRP expression status of a variety of cold treated organizations.It shows the ascites of cell line 3C5 can specifically recognize natural CIRP protein of heart,liver,spleen,lung,kidney,brain,testes and other tissues,and can be combined with CIRP McAb.This proves that the CIRP McAb has a good specificity and binding activity.McAb against CIRP will act as biological reagents to be used in study of the further research of CIRP functions and utilization.

关 键 词：CIRP 原核表达 单克隆抗体 

分 类 号：Q4[生物学—生理学]