Establishing Recombinant Secretory Anti-osteoblastic Sarcoma Single-chain Bifunctional Antibody Gene

Objective: To order to establish recombinant secretory anti-osteoblastic sarcoma single-chain bi-functional antibody gene. Our approach, which has features of both antibody-directed and cell-mediated immunothcrapy, may have application in a gene therapy context. Methods: A single-chain antibody (sFv) gene comprising the linked heavy and light chain variable region derived form a monclonal antibody with high specifity and affinity binding to osteoblastic sarcoma, tumor necrosis factor a (TNF- u ) was fused in frame with sFv gene of downstream by method of PCR. Constructed secretory anti-osteoblastic sarcoma single chain bi-function antibody gene. The sFv-TNF- a gene was then cloned into an expression vector PLXSN. The vector was identified with enzyme digestions for the correct in section and further confirmed by DNA sequence analysis. Result: The expression vector PLXSN- sFv-TNF- a was firstly digested by EcoR 1and Bamlll enzymes, obtained fragments about 930bp were accord with sFv-TNF-a, then was digested by EcoRl and Xhol or Bamill and Xhol enzymes, obtained fragments about 470bp and 460bp were accord with TNF-?and sFv, constructed secretory anti-osteoblastic sarcoma single chain bi-function antibody gene. Conclusions: We are attempting to establish little molecule and mild immunogenicity recombinant single-chain bi-functional antibody, The sFv gene with 5'introduction of leader sequence can express and secrete in eukaryotic cell by method of PCR, the TNF- a gene was linked with sFv gene of downstream, constructed secretory anti-osteoblastic sarcoma single chain bi-function antibody gene. Our next step of experiment, in order to test (he targeted loxicity effect of this bi-functional antibody both in cell level, the sFv-TNF- a gene was shifted into a retroviral vector PLXSN and the recombinant retroviral vector will be transfected into the PA317 amphotropic packaging cells by calcium phosphate method, to establish a stable virus producing cell line. Our approach, which has features of both antibody-directed and cell-mediated immunotherapy, may have application in a gene therapy context.