抗乙型脑炎病毒单抗重链可变区基因的筛选和鉴别  

Screening and Distinguishing Heavy chain Variable region Genes of McAb Against Encephalitis Type B Virus

作　　者：黄华梁[1] 桂进[1] 宋海燕[1] 王艳丽[1] 尚芙蓉[1] 林晴[1] 陈伯权[2] 叶群瑞 吴美英[2] 刘琴芝[2] 杨志兴 

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研究所 [3]黑龙江省应用微生物所

出　　处：《Acta Genetica Sinica》1992年第2期169-176,共8页

基　　金：863资助课题~~

摘　　要：为了制备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抗的51-8杂交瘤细胞株为材料,分离这一单抗的重链可变区基因。杂交瘤细胞的大分子量DNA经Bam HI部分酶切后,以λEMBL-3为载体,构建了总数为2×10~7pfu的基因文库。以抗乙脑病毒单抗重链可变区cDNA为探针,从360000个噬菌斑中筛选出9个阳性斑,经点杂交及Southern杂交证明它们都含有重链可变区基因的片段。进一步以J_(11)探针(含J_3、J_4和重链增强子)对其中4个重组体进行鉴别。经EcoRI酶切后,有3个重组体含有与肝细胞和Sp2/0细胞相同的3.8kb片段,而第4个重组体(λ8a4)没有这一片段,却有一个4.5kb片段,这是在肝细胞和Sp2/0细胞中不存在的。从而证明前3个重组体的插入片段是未经重排的重链可变区基因片段,而λ8a4中的插入片段含有经过重排的功能性可变区基因。这一4.5kb片段不能与含有J_1—J_2的探针杂交,却同时含有V_H、J,或/和J_4和增强子。进一步证明,这是一个功能性的可变区基因。因此,将这一4.5kb片段分离出来之后,在pUC19中亚克隆并作酶切图。为构建抗乙脑病毒的人-鼠嵌合重链基因奠定了基础。In order to prepare human-mouse chimeric antibody against encephalitis type B virus, hy-bridoma 51-8 cells secreting monoclonal antibody against the virus were used as material for isolating heavy-chain variable region gene of the McAb. High molecular weight DNA of the hybridoma cell was partially digested by BamHI, and then constructed a gene library containing 2×107 pfu with λEMBL-3 as vector. With cDNA of heavy-chain variable region of the monoclonal antibody as probe, nine positive plaques were screened from 360 000 plaques, which have been proven that triey contained a fragment of heavy-chain variable region genes by dot hybridization and Southern hybridization. Four recombinants among the nine positive plaques were further distinguished with J11 probe containing Js, J4 and heavy-chain enhancer. After cutting by EcoRl, there was a 3.8 kb fragment in three recombinants as similar to that in liver cell and Sp2/0 cell, but not in the fourth recombinant (λ 8a4) which contained a 4.5 kb fragment that did not present in liver cell and Sp2/0 cell. The results showed that the inserts in former 3 recombinants were non-rea rranged fragment of heavy-chain variable region genes, but the insert in A,Sa4 contained a rearranged functional variable region gene. The 4,5 kb fragment could not be hybridized with a probe containing J1 and J2, but contained VH, J2 or/and J4 and enhancer, that further proved it was a functional variable region gene. Therefore, the 4.5 kb fragment was isolated and subcloned in pUC 19, and its physical map was made. The results lay a foundation for constructing a human-mouse chimeric heavy-chain gene of the antibody against encephalitis type B virus.

关 键 词：乙脑病毒 可变区基因 单克隆抗体 

分 类 号：R392.2[医药卫生—免疫学]