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[Construction of human Gax gene eukaryotic expression vector and its expression in vascular smooth muscle cells]
Aim:
To construct human Gax eukaryotic expression vector of pEGFP-N1-Gax, and observe its expression in the rabbit vascular smooth muscle cells(VSMCs).
Methods:
human Gax cDNA was obtained by polymerase chain reaction(PCR) from pCMV-SPORT6-Gax plasmid. After digested with Nhe I and Xho I, the PCR product was cloned into eukaryotic expression vector pEGFP-N1 of green fluorescent protein(GFP) reported gene encoding green fluorescence protein, and then the recombinant plasmid pEGFP-N1-Gax was transfected into rabbit VSMCs using Sofast after it was identified by restriction enzyme digestion analysis and gene sequencing. Human Gax expression in rabbit VSMCs was detected by examining GFP expression of transfected cells under fluorescence microscope and RT-PCR.
Results:
Agarose gel electrophoresis detection showed that human Gax DNA segment was about 915 bp, which accorded with the expectation. The restriction enzyme digestion analysis and DNA sequencing assays of recombinant vector pEGFP-N1-Gax showed the correct orientation and sequence. The expression of GFP in rabbit VSMCs transfected with pEGFP-N1-Gax was observed by fluorescence microscopy, and the expression of human Gax mRNA was confirmed by RT-PCR.
Conclusion:
The recombinant plasmid pEGFP-N1-Gax is successfully constructed, and expressed positively in rabbit VSMCs, it provide experimental basis to study the effects of Gax gene on cardiovascular disease.
