Preparation Of Anti-Htert Single Chain Fv Antibodies

In this decade, it has been strikingly developed that was the research about single chain Fv(ScFv) in immunity and therapy of tumor. ScFv possessed lots of merits such as low molecular weigh, low immunogenicity, high tissue penetrability, as well as high specilization, so that, compared to monoclone antibody, application of ScFv in tumor diagnosis and treatment might be more promising. Human telomerase is a tumor associated antigen that expression level is up in more than 85% of all kinds of human tumors. A large amount of effort has been focused on the finding of effective telomerase inhibitors that would repress telomerase activity thus limit the growth of human tumor cells. In present experiment, having prepared ScFv of hTERT by reconstruct corresponding monoclone antibody, then its function of tumor repression was investigated by transfection in Lewis lung cancer cell. Using RT-PCR, the variable region gene of anti-hTERT monoclone antibody VL and VH has been cloned from hydridoma cell which could secrete antibody(anti-hTERT) steadily. Then Overlap PCR was performed to connect VL and VH to the aim that producing ScFv gene of hTERT by a linker[(Gly4Ser)4]. The ScFv gene was recombined to prokaryote vector PET19b for expression, that is pET19b-ScFv, which is then transformed to BL21 to be expressed. The product was detected by SDS-PAGE and Western-blot, and its biologic activity of binding to antigen has been obviously detected. In proceeded experiment, ScFv gene was connected to eukaryote vector pEGFP-C1, to construct recombinant, which was then transfected to Lewis lung cancer cell to investigate its effect of tumor repression. Its difference from positive and negative control was analyzed by statistics, the result is that pEGFP-C1-ScFv could repress the growth of tumor cell.