利用人天然Fab段抗体库从肾细胞癌中筛选抗gp96抗体  

SCREENING OF THE FAB ANTIBODIES AGAINST GP96 FROM RENAL CELL CARCINOMA WITH HUMAN NAIVE PHAGE ANTIBODY LIBRARY

作　　者：马小兵[1,2] 畅继武[2] 李慧忠[2] 李成文[2] 

机构地区：[1]华北煤炭医学院病理教研室,唐山063000 [2]天津医科大学第二医院天津市泌尿外科研究所

出　　处：《现代预防医学》2010年第18期3511-3513,共3页Modern Preventive Medicine

基　　金：天津市自然科学基金资助项目(043608411)

摘　　要：[目的]构建人天然Fab段抗体库,并用从肾细胞癌组织中提取纯化的gp96,并以此来筛选抗gp96特异性抗体,为恶性肿瘤的免疫治疗提供新方法。[方法]取健康新鲜血液50ml,分离淋巴细胞,提取总RNA,经逆转录合成cDNA,设计多对引物,以PCR扩增抗体轻链和重链Fd段cDNA。轻链PCR产物和噬粒载体pComb3经SacⅠ和XbaⅠ酶切,由T4DNA连接酶连接,电穿孔转化感受态大肠杆菌XL1-Blue,扩增后提取噬粒,构建轻链库。轻链库和重链Fd段PCR产物经XhoⅠ和SpeⅠ酶切,以同样方法连接后转化XL1-Blue,加入辅助噬菌体VCSM13,产生噬菌体抗体全库。提取噬粒酶切鉴定轻链库和全库有无插入片段。分别以噬菌体全库和提取的噬粒为模板,以轻、重链不同引物组合进行PCR扩增。应用亲和层析和离子交换层析从肾细胞癌组织中提取纯化gp96,并以此为抗原对构建的抗体库进行3轮筛选。[结果]提取的淋巴细胞总RNA及逆转录合成的cDNA质量好,部分重链Fd片段、部分κ轻链和λ轻链cDNA得到扩增。Fab库容为1.2×106,酶切和PCR显示有插入片段。用从肾细胞癌组织中提取出的gp96进行3轮筛选,抗体库得到71.5倍的富集。[结论]本研究构建的人天然Fab段噬菌体抗体库可为进一步筛选特异性抗体,用于免疫治疗打下基础。[Objective] To construct a naive human Fab fragment phage display library,gp96 purified from the tissue of renal cell carcinoma to screen the anti-gp96 antibodies and provide a platform for human antibody preparation and a basis for new therapy of the malignant tumors.[Methods] With lymphoprep,peripheral blood lymphocytes were isolated from 50ml of blood,which was obtained from a healthy blood donor.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR with variable regions 5＇ and 3＇ primers of heavy and light chain,and the amplifica-tion products were ligated into the phagemid vector pComb3,then the ligated sample was transformed into competent E.coli XL1-Blue by Electroporation.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage anti-body Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as SacⅠ,XbaⅠ,SpeⅠand Xho Ⅰ,and the phage antibody Fabs or phagemids abstracted from amplified E.coli were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment.The gp96 purified from the renal cell carcinoma tissue by affinity chromatography on concanavalin-A sepharose and DEAE-sepharose ion exchange chromatography were utilized as antigens to pro-cess three rounds of panning to the original Fab antibody library.[Results] The quantity of total RNA and cDNA were quali-fied.By combination of light chain and heavy chain genes,an antibody library containing 1.2×106 clones was obtained,and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids.The gp96 protein was obtained from renal cell carcinoma tissue.After having been panned by gp96,the original an-tibody library gained enrichment in 71.5 times.[Conclusion] Utilizing the technology of phage surface display,special antibody can be gained from the human naive Fab phage display library,which can be used as a new therapy for tumors.

关 键 词：噬菌体抗体库 抗体FAB段 GP96 肾细胞癌 

分 类 号：R737.11[医药卫生—肿瘤]