人巨细胞病毒pp65原核表达载体构建及表达产物对特异性免疫应答的诱导  

The Construction of Human Cytomegalovirus pp65 Prokaryotic Expressing Vector and the Inducement of Specific Immune Response

作　　者：赵捷[1] 闻良珍[1] 陈素华[1] 凌霞珍[1] 

机构地区：[1]华中科技大学附属同济医院妇产科

出　　处：《中华围产医学杂志》2003年第2期109-112,共4页Chinese Journal of Perinatal Medicine

摘　　要：目的　构建人巨细胞病毒 pp6 5原核表达载体 ,研究纯化后的重组 pp6 5蛋白诱导特异性免疫应答的作用及其特异性。　方法　采用体外 DNA重组技术 ,构建 pp6 5原核表达载体并诱导其表达 ,通过亲和层析对表达产物进行纯化和经 Westen blot鉴定 ;用 MTT法检测重组蛋白对人外周血单个核细胞 (peripheral blood m ononuclear cell,PBMC)的激活、增殖作用 ,检测激活淋巴细胞对受 HCMV感染的人胚肺成纤维细胞 (human em bryo lung fibroblast,HEL )的杀伤作用。　结果　成功构建 pp6 5原核表达载体并诱导其高效表达 ,纯化后的 pp6 5蛋白能促进 PBMC激活和增殖 ,增殖的细胞可特异性地杀伤受 HCMV感染的 HEL细胞。　结论　经重组 pp6 5蛋白激活增殖的淋巴细胞 ,可特异性杀伤受 HCMV感染细胞 ,对 HCMV感染的免疫治疗具有十分重要的作用。Objective To construct human cytomegalovirus pp65 prokaryotic expressing vector and induce specific T lymphocyte immune response with recombinant pp 65 protein of human cytomegalovirus(HCMV) to observe the effect of CTL on infected HEL cells. Methods The whole cDNA of HCMV pp65 was amplified by PCR and inserted into prokaryotic expressing vector pRSET by gene engineer technique. The product was purified by affinity chromphotography and identified with Western blot after that recombinant plasmid was expressed by the induction of IPTG. With MTT technique, we observed the stimulating and proliferating effect of recombinant HCMV pp65 protein on PBMC in vitro. Cytotoxicity of PBMC on HCMV-infected HEL cells was detected. Results The pp65 prokaryotic expressing vector was successfully constructed and could express in engineering bacteria DE3. High dose of pure recombinant protein was acquired and had been identified. The rhHCMV pp65 protein can activate PBMC and cause the proliferation of it in vitro. The proliferated PBMC have the specific cytotoxicity to HEL cells infected by HCMV. Conclusions The acquired recombinant HCMV pp65 protein could induce specific T cells immune response in vitro to kill the HCMV infected HEL cells. And it is very important for the immune therapy of the HCMV infections.

关 键 词：巨细胞病毒 磷蛋白类 病毒基质蛋白质类 单核白细胞 淋巴细胞转化 

分 类 号：R392[医药卫生—免疫学]