Cloning And Sequencing Of Variable Region Genes Of Rat Monoclonal Antibody Against HTV, Expression Of Rat-human Chimeric Heavy Chain Antibody Specific For HTV In Mouse Myeloma Cell And Construction Of ScFv Gene Displayed On The Surface Of Phage

We have used acid-Guanidine Thiocyanate method to extract total RNA of rat hybridoma cells, which secreted monoclonal antibody against HTV, then synthesized first strand cDNA. We have designed a set of oligonucleotied primers to amplify the cDNA of rat immunoglobulin variable- region genes by polymerase chain reaction. Here we have applied the technique to clone and sequence the varaible domain of monoclonal antibody. The results show that the lengths of light and heavy variable-region of this antibody are 351 and 360 base pairs respectively.The heavy variable- region gene of rat monoclonal antibody specific for HTV, was inserted into pTZV2F7BII to get pTZRatVHBII, then recombined the recombinant with immunoglobulin heavy chain enhancer and vector pSVneo, a new vector pSV-VE was acquired. At last a chimeric plasmid pSVHgptRatVHHur3 was constructed based on the mouse immunoglobulin heavy variable-region and Human immunoglobulin constant-region gene. It possesed all the expression elements. The recombinant plasmid containing a chimeric gene encoding the human CH domain and the rat VH domain with specificity for HTV was introducted into a mouse myeloma cell line (J558L) by electroporation. Three positive clone were obtained and...