hCGβ-CTP37多聚体的表达、纯化及其多克隆抗体的制备  

Expression, Purification, and Polyclonal Antibodies Production of hCGβ-CTP37 Polymerides

作　　者：喻光荣[1] 蒋俶[2] 冯文华[1] 沈卫英[2] 蒋文珏[2] 申庆祥[2] 

机构地区：[1]复旦大学上海医学院,上海200032 [2]上海市计划生育科学研究所,上海200032

出　　处：《中国生物工程杂志》2005年第11期21-26,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(30472087);上海市科技攻关重大项目(03DZ19229)

摘　　要：目的:研究重组hCGβCTP37(CTP)多聚体编码基因在大肠杆菌BL21中的表达及表达产物多克隆抗体的制备。方法:分别将3、4个CTP编码基因通过串联的方式连接起来组成多聚体编码基因,进而将多聚体基因亚克隆入载体pGEX4T2中获得含目的基因的表达质粒pGEX4T2(CTP)n(n=3,4)。表达质粒转入大肠杆菌BL21,目的蛋白在IPTG诱导下进行表达。表达产物经谷胱甘肽转硫酶(GST)亲和层析柱纯化、凝血酶酶解去除GST后,获得多肽CTP3和CTP4。将其用于免疫家兔以制备多克隆抗体,并用ELISA法检测抗体滴度。结果:菌株经IPTG诱导后产生了大量可溶性的融合蛋白GSTCTPn(n=3,4),SDSPAGE分析表明融合蛋白GSTCTP3和GSTCTP4的表观分子量分别为38.0kDa和42.2kDa,纯化后的融合蛋白的纯度达到了90%以上;纯化的融合蛋白经牛凝血酶酶解,利用制备型SDSPAGE纯化,回收获得的多肽CTP3和CTP4的纯度均大于80%;将多肽CTP3和CTP4多次免疫家兔后,ELISA检测结果证实产生了滴度较高的多克隆抗体。结论:CTP多聚体能刺激家兔产生特异性抗体,这提示CTP多聚体有可能作为避孕疫苗或肿瘤疫苗的免疫原。Objective ： To study the expression of hCGβ-CTP37 polymeride in the E. coli BI21 （DE3） and the antibody production of the expressed products. Methods： Three or four CTP cDNA were strung to construct CTP polymeric cDNA, and two expression plasmids, pGEX-4T-2-（CTP）n（n = 3,4）, were constructed by inserting CTP polymeric cDNA into plasmid pGEX-4T-2 containing GST gene. After identified by DNA sequencing, the two expression plasmids were introduced into E. coli BL21 cells and the fused proteins were expressed in the expression strains induced with IPTG. Purified by GST Sepharose 4B Column, the expressed products were digested by thrombin protease to obtain the peptides of CTP3 and CTP4. Then, the rabbits were immunized with the purified CTP3 and CTP4 which were isolated by SDS-PAGE to produce antibody, and the titer of these antibodies was investigated with ELISA. Results： The expression strains expressed a substantial amount of soluble fused proteins GST-CTP3 and GST-CTP4 induced by IPTG, and SDS-PAGE analysis revealed the fused proteins GST-CTP3 and GST-CTP4 had apparent molecular weight of 38.0kDa and 42.2kDa respectively. After purified, the fused proteins had the purity of more than 90% and were digested by thrombin protease. After purified by productive SDS-PAGE, the peptides of CTP3 and CTP4 had the purity of more than 80%. Then, the purified CTP3 and CTP4 were used to immunize rabbits three times, and polyclonal antibodies were obtained from the serum of immunized rabbits. ELISA and Western blot analysis showed the antibody specificity to CTP3 and CTP4. Moreover, ELISA analysis indicated that the titer of anti-CTP4 antibody was higher than that of anti-CTP3 antibody. Conclusion： CTP polymeride can stimulate the rabbits to produce the specific antibodies, which suggested the possibility of CTP polymeride as the immunogen for anti-hCG contraception vaccine or cancer vaccine.

关 键 词：hCGβ-CTP37 大肠杆菌基因表达抗体 

分 类 号：Q786[生物学—分子生物学] TQ464.56[化学工程—制药化工]