[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.
Shang
F R
FR
Huang
H L
HL
Song
H Y
HY
Chen
H
H
Yang
Z X
ZX
D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000235:genetics*; D002002:Brucella / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D004247:DNA