CONSTRUCTION AND IDENTIFICATION OF EUKARYOTIC EXPRESSING PLASMID pVAX1-hIL-10

Objective To clone the gene of human interleukin-10(hIL-10),so as to construct its eukaryotic expressing plasmid,and to detect its expression in vitro COS7 cells.Methods The gene of hIL-10 was amplified from human peripheral blood by reverse transcriptase-polymerase chain reaction(RT-PCR), and then inserted it into cloning vector pMD18-T,and eukaryotic expressing vector pVAX1 respectively.Recombinant plasmids were transformed into E.coli DH5α and screened,and identified by PCR,restriction enzyme digestion and DNA sequencing.The recombinant expressing plasmid pVAX1-hIL10 was transfected into COS-7 cells by cationic liposome.Then detected the expression of the interesting gene hIL-10.Results The recombinant plasmid pMD18T-hIL10 and pVAX1-hIL-10 were successfully constructed,and the correct sequence of hIL-10 was identified by PCR,restriction enzyme digestion and DNA sequencing.The recombinant expressing plasmid pVAX1-hIL-10 was successfully transfected into COS-7 cells and it could be effectively expressed,which was also testified by Western blot.Conclusion The recombinant eukaryotic expressing plasmid of hIL-10 is successfully constructed and effectively expressed in COS-7 cells,and it may be useful for further research.