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[Screening and distinguishing heavy chain variable region genes of McAb against encephalitis type B virus]
In order to prepare human-mouse chimeric antibody against encephalitis type B virus, hybridoma 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 x 10(7) pfu with lambda EMBL-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 they 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 J3, J4 and heavy-chain enhancer. After cutting by EcoR1, 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 (lambda 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-rearranged fragment of heavy-chain variable region genes, but the insert in lambda 8a4 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, J3 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.(ABSTRACT TRUNCATED AT 250 WORDS)
In order to prepare human-mouse chimeric antibody against encephalitis type B virus, hybridoma 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 x 10(7) pfu with lambda EMBL-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 they 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 J3, J4 and heavy-chain enhancer. After cutting by EcoR1, 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 (lambda 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-rearranged fragment of heavy-chain variable region genes, but the insert in lambda 8a4 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, J3 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.(ABSTRACT TRUNCATED AT 250 WORDS)