[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)
Huang
H
H
Institute of Genetics, Academia Sinica, Beijing.
Gui
J
J
Song
H
H
Wang
Y
Y
Shang
F
F
Lin
Q
Q
Chen
B
B
Ye
Q
Q
Wu
M
M
Liu
Q
Q
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000914:Antibodies, Viral / Q000235:genetics*; D004668:Encephalitis Viruses / Q000276:immunology*; D015723:Gene Library; D005803:Genes, Immunoglobulin; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region