人源性抗前列腺特异性膜抗原单链抗体的基因构建、表达及鉴定  

Construction,expression,and identification of the gene of human anti-prostate specific membrane antigen single-chain antibody

作　　者：苏燕胜 高云 付晓亮[2] 王东[2] 王倩云 刘娜[3] 贾红兵 秦卫军[4] 温伟红[5] 王禾[2] 

机构地区：[1]解放军323医院泌尿外科,陕西西安710054 [2]第四军医大学唐都医院泌尿外科,陕西西安710032 [3]西安交通大学第二附属医院药剂科,陕西西安710004 [4]第四军医大学西京医院泌尿外科,陕西西安710032 [5]第四军医大学免疫教研室,陕西西安710032

出　　处：《中华男科学杂志》2014年第12期1063-1067,共5页National Journal of Andrology

基　　金：国家自然科学基金(30973000)~~

摘　　要：目的:分别构建人源性抗前列腺特异性膜抗原(PSMA)单链抗体(sc Fv)/鱼精蛋白截短体(tp)、sc Fv/弗林蛋白酶识别位点(Fdt)/流感病毒融合肽结构域(HA2)/tp融合蛋白基因。利用原核表达体系表达、纯化并检测sc Fv、sc Fv-tp、sc Fv-Fdt-HA2-tp融合蛋白的活性。方法:采用PCR的方法,扩增融合基因sc Fv、sc Fv-tp、sc Fv-Fdt-HA2-tp,将获得的基因克隆入原核表达载体p ET28,在大肠杆菌BL21中表达,表达产物经SDS-PAGE和Western印迹鉴定,通过Ni2+-NTA螯合层析纯化。ELISA分析融合蛋白抗原亲和活性。结果:成功构建了人源性抗PSMA融合基因,经IPTG诱导后在M15中以包涵体形式表达。表达的目的蛋白均能与PSMA抗原结合。结论:融合蛋白具有结合抗原的活性,为靶向递送siRNA的研究奠定了基础。Objective： To construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen （PSMA） protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins. Methods： The fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET＇28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni^2＋ -NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA. Results： The human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen. Conclusion： Fusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.

关 键 词：前列腺特异性膜抗原 单链抗体 融合蛋白 靶向输送 

分 类 号：R392.1[医药卫生—免疫学]