Construction of eukaryotic expression vectors of mouse HEPC1 and HEPC2 genes and expression in NIH3T3 cells

Objective To construct the eukaryotic vectors of mouse HEPC1 and HEPC2 genes and observe their expressions in NIH3T3 cells lines. Methods To obtain HEPC1 and HEPC2 complete cDNAs, the total RNA was isolated from liver of mouse and cDNAs were synthesized by RT-PCR. The specific amplification products were cloned into pcDNA3 and then were identified by restriction enzymes digestion and sequencing analysis. The recombinant plasmids pcDNA3-HEPC1 and pcDNA3-HEPC2 were respectively transfected into NIH3T3 cells by lipofectin. The positive clones of the transfected cells were obtained by G418 screening, and their expressions were analyzed by RT-PCR and dot-ELISA. Results The expected gene fragments were obtained by RT-PCR. The recombinant plasmids were identified to contain target fragments by restriction enzyme digestion analysis. DNA sequencing showed the sequence of HEPC2 gene had no difference with the GenBank reports while the sequence of HEPC1 gene had a base difference from the GenBank reports, but it did not affect the coded amino acid. The HEPC1 and HEPC2 were successfully expressed in transfected NIH3T3 cells. Conclusion Eukaryotic expression vectors pcDNA3-HEPC1 and pcDNA3-HEPC2 were constructed successfully. HEPC1 and HEPC2 were successfully expressed in the transfected NIH3T3 cells, which laid the foundation for further studies on the biological functions of HEPC1 and HEPC2 and their mechanisms.