Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells.
OBJECTIVE
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 immunofluorescence staining.


CONCLUSIONS
The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.
Jiang
Yong-Fang
YF
Center for Liver Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, Hunan Province, China. jiangyongfang@hotmail.com
He
Yan
Y
Gong
Guo-Zhong
GZ
Chen
Jun
J
Yang
Chun-Yan
CY
Xu
Yun
Y
D000818:Animals; D001483:Base Sequence; D023201:CD40 Ligand / Q000235:genetics*; D006528:Carcinoma, Hepatocellular; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D017931:DNA Primers; D005260:Female; D005786:Gene Expression Regulation / Q000276:immunology; D008113:Liver Neoplasms; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000276:immunology
D017931:DNA Primers; D023201:CD40 Ligand