Genomic DNA fragment encoding antibody variable region prodn. - by attaching primers to hybridoma DNA then subjecting to polymerase chain reaction, for constructing genes encoding chimeric antibodies

摘要

Isolation of a DNA fragment (I) which encodes the variable region of an antibody (Ab) comprises (1) isolating DNA from a hybridoma cell line which secretes the required Ab; (2) converting the DNA to single-strand form, which is then incubated with 2 oligonucleotide primers; primer (1) has a region complementary to a sequence of at least 10 nucleotides from the 5'-untranslated region of the template strand of the Ab gene, while primer (2) contains a region complementary to a sequence of at least 10 nucleotides of the 3'-untranslated region of the relevant J-region of the non-template strand; (c) reacting with DNA polymerase and dNTP to elongate the primers, forming a double-stranded fragment between the 2 primers; (d) denaturing the prod. to single strand form which is reacted again with (1) and (2), and the entire procedure repeated as often as needed.