[Synthesis and cloning of V gamma 3 cDNA of monoclonal antibody]

With the guanidinium isothiocyanate method, total RNA was isolated from hybridoma cells that secrete monoclonal antibody against Brucella melitenses. Poly (A)+ RNA was obtained by oligo (dT)-cellulose affinity chromatography. Reverse transcriptase reaction was performed with a primer 3'A-T-A-G-G-T-G-A-C-C 5' that is complement to the codons of No. 122-125 amino acid residues in 5' terminus of constant region. The size of synthesized ds-cDNA is about 300bp, that is consistent with the length of variable region genes of heavy chain. The ds-cDNA was inserted into plasmid pUC19 with dC: dG tailing method, and the inserted plasmid was used to transform E. coli HB101. It has been proved that the insert was a variable region gene of heavy chain by clone hybridization in situ, size of insert and Southern blot.