人天然噬菌体抗体库的构建及抗gp96抗体的初步筛选

Construction of human naive phage antibody library and primary screening of the Fab antibodies against gp96

目的构建人天然Fab段抗体库，并用从膀胱尿路上皮癌组织中提取纯化的gp96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4个健康献血员新鲜血液各200ml，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Sac Ⅰ和Xba Ⅰ酶切，由T4 DNA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Xho Ⅰ和Spe Ⅰ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膀胱癌组织中提取纯化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全库库容达6．6×10^6，酶切和PCR显示有插入片段。用从膀胱尿路上皮癌组织中提取出的gp96进行3轮筛选，抗体库得到68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、肿瘤免疫治疗打下基础。

Objective To construct a naive human Fab fragment phage display library,from which the anti- gp96 antibodies may be panned by the gp96 purified from the tissue of urothelial carcinoma in the urinary bladder and provide a basis to new therapy for the malignant tumors. Methods Peripheral blood lymphocytes were isolated from 800 ml of blood ,which was obtained from four healthy blood donors. The heavy chain Fd and light chain eDNA synthesized from the total RNA of lympboeytes were amplified by PCR with variable regions 5＇ and 3＇ primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E. eoli XL1-Blue by eleetroporation. The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs. The phagemids abstracted from amplified E. coli were cut with endonucleases such as Sac Ⅰ , Xba Ⅰ , Xho Ⅰ and Spe Ⅰ , and both the phage antibody Fabs and 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 urothelial carcinoma tissue of the bladder by affinity chromatog- raphy on concanavalin-A sepharose and DEAE-sepharose ion exchange chromatography were utilized as antigens to process three rounds of panning to the original Fab antibody library. Results The quantity of total RNA and cDNA were qualified. By combination of light chain and heavy chain genes ,an antibody library containing 6.6 × 10^6 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 urothelial carcinoma tissue in the urinary bladder. After having been panned by gp96, the original antibody library gained enrichment by 68 times. Conclusion Utilizing the technology Of phage surface display, specific antibody can be gained from the human naive Fab phage display library, which can be used for immunological therapy for tumors.