Construction of eukaryotic expressing plasmid of human CD63 gene and its expression and location in TCA8113 cells

Objective Toconstruct an eukaryotic expressing plasmidof human CD63 gene and observe its expression and location in TCA8113 tongue squamous cell carcinoma cell line. Methods Total RNA was extractedfromTCA8113 cells. The CD63 gene was amplifiedby PCR methodand subclonedtopcDNA3.1 (+) plasmid. The recombinant plasmids were transformedintoE.coli DH5α and screenedby restriction enzyme digestion and DNA sequencing. Then, pcDNA3.1-CD63 plasmids were transfectedintoTCA8113 cells. Western blot was usedtodetect the expression of protein, the subcellular location between endogenous CD63 and pcDNA3.1-CD63 in TCA8113 cells was detectedand comparedby indirect immunofluorescence staining. Results The recombinant eukaryotic expressing plasmid, pcDNA3.1-CD63, was successfully constructed. The expression of recombinant plasmidin TCA8113 cells was provedby Western blot. Andthe indirect immunofluorescence staining showedthat the location of pcDNA3.1-CD63 protein was similar with endogenous CD63 protein, and they both locatedin the outer membrane of TCA8113 cells. Conclusion The fusion protein pcDNA3.1-CD63 can be successfully expressedin TCA8113 cells and locatedin the outer membrane.