抗乙型脑炎病毒单克隆抗体轻链可变区基因的分离和鉴别  

CLONING AND IDENTIFICATION OF LIGHT-CHAIN VARIABLE REGION GENE OF MONOCLONAL ANTIBODY AGAINST ENCEPHALITIS TYPE B VIRUS

作　　者：黄发灿[1] 黄华梁[1] 熊曦明[2] 桂进[1] 熊伟[1] 林晴[1] 宋海燕[1] 陈伯权[2] 

机构地区：[1]中国科学院遗传研究所,北京100101 [2]中国预防医学科学院病毒学研究所

出　　处：《病毒学报》1993年第1期23-29,共7页Chinese Journal of Virology

基　　金：国家863高科技生物技术领域资助

摘　　要：为了构建抗乙型脑炎病毒的人—鼠嵌合抗体,以分泌抗乙型脑炎病毒单克隆体的51-8杂交瘤细胞株为材料,以J_κ和V_κ为探针,从λEMBL-3为载体构建的基因文库中分离有功能性的轻链可变区基因。经3次复筛得到19个阳性噬菌斑。将阳性噬菌斑的DNA重组体和小鼠肝DNA分别用BamH I酶切后进行分析,在分析的8个重组体中,其中5个含有2.8kb片段,而肝DNA含有8.9kb、5.6kb、3.6kb及3.1kb4个片段,没有2.8kb片段。由于肝DNA的4个片段是未经重排的种系基因,因此可以推断重组体中的2.8kb片段是经过重排的轻链可变区基因。为了进一步鉴定,又从中选出3个含有2.8kb片段的重组噬菌斑,以V_κ和J_κ作探针分别进行点杂交,结果都为阳性,进一步说明了2.8kb片段是经过重排的轻链可变区基因。将这一2.8kb片段克隆到pUC19中,得一重组子,命名为pVLD24。利用pCR扩增技术从pVLD24中扩增出预期的334 bp轻链V基因。综合证实了所鉴别得到的功能性轻链可变区基因是完整的,并且用不同的限制性内切酶绘制了这一功能性基因的酶切图谱。In order to prepare human-mouse chimeric antibody against encepha-litis type B virus, hybridoma 51-8 celis secreting monoclonal antibody against the virus were used as material for isolating light-chain variable region gene of McAb. With JK as probe, nineteen positive plaques were screened from 300,000 plaques in a gene library containing 2×107 pfu with AEMBL-3 as vector. Eight recombinants DNA, mouse liver DNA and Sp2/0 myeloma cell DNA were cut by BamHI respectively and Southern hybridization and gene rearrangment analysis were carried out. Five recombinants were proven to contain a 2.8kb fragment among eight recombinants analysed, while mouse liver DNA contained 8.9kb, 5.6kb, 3.6 kb and 3.1kb fragments without 2.8kb fragment. As fragments of liver DNA are unrearranged germline genes, it is probable that the 2.8kb fragment is rearranged light-chain variable region gene. Three recombinants containing 2.8kb fragment were further analysed by dot hybridization with JK and VK as probes, further confirming that the 2.8 kb fragment was rearranged light-chain variable region gene. Therefore, the 2.8kb fragment was isolated and subcloned in pUCl9, and a recom-binant was obtained, designated as pVLD24. Using PCR technique, a 334 bp light-chain V gene was amplified from pVLD24, comprehensively proving that the functional light-chain variable region gene was obtained. Its physical map was prepared by restriction enzymes.

关 键 词：乙型脑炎病毒 单克隆抗体 轻链 

分 类 号：R373.31[医药卫生—病原生物学]