The Construction And Expression Of Anti-lung Single-chain Antibody Conjugated With Green Fluorescent Protein Gene

Use of the green fluorescent protein(GFP) of Aequorea victoria as a reporter has provided sensitive new approaches for real time monitoring in living cells. More and more reports have demonstrated that when GFP fused to target antibody, the host cell can express bifunctional fusion protein, which has the fluorescent character of GFP and the antibody activity. As an alternative to immunofluorescence microscopy, the expression of GFP gene fusions has been used for tumor monitoring, high-throughput screening drugs or researching the function of the acceptor of certain cell factor.In this paper, a expression vector that contains GFP gene and anti-lung cancer ScFv gene is construct. First, the variable region of heavy chain and light chain is cloned from cDNAs through PCR. Then, VL gene is amplified and modified with linker primer, Equal mole of VH and modified VL are then subjected to SOE ligation and ScFv gene is amplified with Vhbak and Vlfor. Sequencing results indicate that no point mutation and frame-shifting mutation are brought about during PCR amplification. The GFP gene is cloned into pPROEX HTb plasmid between the Nco I/Xba I sites. After modified by PCR using sfvl and vlfor primers so that the amplified fragment contains Bgl II /Xho I sites, the PCR product and the recombinant vector are digested with Bgl II +Xho I , the modified scfv gene is inserted into the C-terminal of GFP gene between Bgl II /Xho I sites, then transforming the recombinant vector into E.coli.Compared to the negative bacterial, the constructed plasmid gave a high level of expression similar to the anticipated protein after IPTG induction. It appears two sharp peaks through the purification of Ni-NTA resin, the analysis of SDS-PAGE shows that one of the molecule weight of the fusion protein is similar to the target protein. The host cells fluoresce bright green under 396nm wavelenth, it means the gene has expressed in the cells, but whether the target protein has the biological function needs further research.