Construction And Expression Of SLC-anti CEA ScFv Bifunctional Antibody Fusion Protein

Secondary lymphoid tissue chemokine (SLC) is chemotactic for T lymphocytes and mature DC and could trigger T cell homing and SLC showed definited T-cell dependent antitumor activities. CEA (carcinoembryonic antigen) molecule is a wide tumour symbol locating on the surface of malignant cells or in the blood of some kinds of cancer patients. So it is considered as a tumor associated antigen (TSA). Anti-CEA antibodies are often used as " homing devices", with improved specificity, delivering cytotoxic radionuclides, chemotherapeutic agent, toxins or prodruges, to kill CEA-bearing tumor cells in vivo.Cytokine-antibody fuse protein can attract cytokine to the focus of tumour and can effectively control tumour growth in animal model. It is an effective specific immunotherapy of tumor which have the specific of antibody and mutifuction of cytokine .The nucleotide sequences that coding SLC and anti-CEA ScFv had been cloned into the vector PALM of which the muticlon location had been changed and fit for expressing bispecific-single-chain antibody. A fusion protein of SLC-anti CEA ScFv had been consetructed. In this study, the coding sequence of SLC-anti CEA ScFv was abtained by PCR from the expression vector PALMSLC-anti CEA ScFv , and then was disposed by enzymy and cloned into the vector of PTMFand PTFRSM. The results of PCR and sequence determining showed the expression vectors of PTMFSLC-anti CEA ScFv and PTFRSMSLC-anti CEA ScFv were constructed successfully. Analysis of SDS-PAGE and Western blotting showed the fusion protein was expressed in different vector. The expression condition was fished out in different vector from inducement time and temperature. The fusion protein was expressed almost as a format of inclusion bodies in the vector of PALM and expression quantity was highest. The protein expressed in PTFRSM was mostly soluble which proved that FkpA could markedly improve the soluble quantity of the expression protein. The expression protein were purified with Ni-NTA chelate agarose ,purification rate could arrive 90%.SLC-anti CEA ScFv was capable of binding to T cell and human colon cancer as detected by ELISA analysis of Jurkat cells and SW116 cells respectively. Freshly isolated peripheral blood leukocyte showed migration toward the purified protein SLC-anti CEA ScFv, the chamotactic index was maximal at 0.225 ug/ml -1.65ug/ml. The results of ELISA and chemotaxis assay indicated that a functional fusion protein was obtained, and was proved having potential anti-tumour activity.