基因工程鼠Muc1-MBP融合蛋白的制备及其免疫活性测定  

Preparation and immunological activity analysis for the gene engineering mouse Muc1-MBP fusion protein

作　　者：方芳[1] 马吉春[1] 高航[1] 赵小霞[1] 周静[1] 宋献美[1] 柳忠辉[1] 台桂香[1] 

机构地区：[1]吉林大学白求恩医学院免疫教研室,长春130021

出　　处：《中国免疫学杂志》2009年第3期251-254,270,共5页Chinese Journal of Immunology

基　　金：教育部留学人员启动基金;吉林大学创新基金资助

摘　　要：目的：制备鼠Muc1-MBP融合蛋白,探讨其免疫活性。方法：将pMAL-Muc1转化大肠杆菌,通过IPTG诱导、SDS-PAGE和Western blot鉴定Muc1的表达,Amylose resin亲和层析纯化Muc1-MBP。将Muc1-MBP融合蛋白免疫小鼠,采用ELISA方法检测小鼠血清Muc1特异性抗体的水平;MTT法测定小鼠抗Muc1特异的CTL活性;ELISPOT法测定脾脏淋巴细胞IFN-γ的分泌。结果：获得稳定表达Muc1的菌株,制备了分子量67 kD较纯的Muc1-MBP融合蛋白。Muc1-MBP融合蛋白免疫小鼠产生高效价Muc1特异抗体,效价在8 000~16 000之间,Muc1-MBP融合蛋白可诱导CTL杀伤活性,对人乳腺癌MCF-7和小鼠Lewis肺癌LLC1靶细胞的杀伤率分别为（74.5±5.9）%和（60.5±10.4）%,与对照组比P〈0.05,差异显著;Muc1-MBP融合蛋白免疫可诱导脾脏淋巴细胞分泌IFN-γ,特异性活化Th1细胞。结论：成功制备重组鼠Muc1-MBP融合蛋白,其不仅能诱导特异体液免疫应答,而且可诱导细胞免疫应答,尤其能诱导对人类MCF-7细胞较小鼠Lewis肺癌LLC1细胞更加强烈的CTL杀伤活性,提示异种同源蛋白疫苗具有比同源蛋白疫苗更好的抗肿瘤作用。Objective：To prepare the mouse Muc1-MBP fusion protein and to investigate its immunological activity.Methods：pMAL-Muc1 was transformed into E.coil,Muc1-MBP expression was induced by IPTG and identified by Western blot.The Muc1-MBP protein was purified by Amylose resin affinity chromatography.Then female C57BL/6 mice were immunized with Muc1-MBP fusion protein.Muc1-specific antibody was detected by ELISA.CTL cytotoxicity was detected by MTT.The production of IFN-γ of spleen lymphocytes was assessed by ELISPOT.Results：The strain of stable expression Muc1 was obtained.Purified Muc1-MBP fusion protein with molecular weight of 67 kD was prepared.Muc1 specific antibody was produced in the mice immunized with Muc1-MBP protein,and the titer was ranged from 8 000 to 16 000.Muc1-MBP fusion protein could activate specific CTLs.The killing effect of CTLs to MCF-7 and LLC1 target cells was（74.5±5.9）% and（60.5±10.4）% respectively,which was significantly increased compared with the control group（P〈 0.05）.Lymphocytes of immunized with Muc1MBP groups could induce IFN-γ production and active specific Th1 cells.Conclusion：Muc1-MBP fusion protein can induce not only specific humoral immunoresponse,but also cellular immunologic response,which especially elicit stronger cytotoxicity of CTL to MCF7 cells than to LLC1 lung cells.Our results suggests that the antitumous effect of heterogenetic protein vaccine was better than homologous protein vaccine.

关 键 词：鼠Muc1 Muc1-MBP融合蛋白 CTL活性 异种同源蛋白 肿瘤疫苗 

分 类 号：R392.33[医药卫生—免疫学]