Efficient recombination of Lym-1 scFv gene using multiple doubly-restricted DNA fragments.
In order to improve radioimmunotherapy of lymphoma, a Lym-1 single-chain antigen-binding (scFv) protein molecule was produced. Because the commonly used polymerase chain reaction (PCR) method frequently causes unexpected mutations, we developed a non-PCR method for scFv gene assembly. The method involved a stepwise linkage of doubly-restricted DNA fragments and re-digestion of the resultant concatamers. Using this strategy, the Lym-1 scFv expression gene was readily constructed without mutations. The recombinant gene was cloned into an expression vector and scFv protein was expressed. The method can be used for other genes or DNA recombination.
Shi
X B
XB
Department of Internal Medicine, University of California, Davis, Medical Center, Sacramento, USA.
Gumerlock
P H
PH
Kroger
L
L
DeNardo
G L
GL
DeNardo
S J
SJ
D000911:Antibodies, Monoclonal / Q000235:genetics*; D058846:Antibodies, Monoclonal, Murine-Derived; D004274:DNA, Recombinant / Q000138:chemical synthesis* / Q000302:isolation & purification; D015246:Deoxyribonuclease EcoRI / Q000494:pharmacology; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D005803:Genes, Immunoglobulin; D005813:Genes, Synthetic; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D004274:DNA, Recombinant; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; C410509:Lym-1 monoclonal antibody; D015246:Deoxyribonuclease EcoRI; C027908:endodeoxyribonuclease DdeI; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases