AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the sp...AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.展开更多
基金Supported by the Public Health Department Foundation of Hunan province, China, No. Y02-42
文摘AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.
