Objective:To construct the mammalian CO-expression plasmid pBudCE4.1-LMP-1-LMP-3

and to detect the expression of the plasmid in vitro.Methods:Fragments of LMP-1 gene and Fragments of LMP-3 gene were gained from the Company

and were constructed respectively into the plasmid vector Puc57

The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes.fragments of LMP-1 gene were constructed firstly into the plasmid vector pBudCE4.1

fragments of LMP-3 gene were constructed into the plasmid vector pBudCE4.1-LMP-1 after iden tification with nucleotide sequencing and enzymes.MSCs cell line was transfected with this Co-expression plasmid using lipofectin reagent.according to the transfect situation

the MSCs were divided into 5 groups

the non-transfected group(Group A)

the group transfected by empty vector(Group B)

the group transfected by LMP-1(Group C)

the group transfected by LMP-3(Group D)and the group transfected both LMP-1 and LMP-3(Group E).the expression of LMP-1 and LMP-3 were detected by RT-PCR and Western blot technique.Results:The plasmids Puc57-LMP-1、Puc57-LMP-3 and pBudCE4.1-LMP-1-LMP-3 were obtained successfully and verified by nucleotide sequencing and enzymes.After transfection with this mammalian Co-expression plasmid

the LMP-1 and LMP-3 molecules were expressed in MSCs cells.The results of RT-PCR and Western Blot were measured with the grey value.To the expression of mRNA and protein of LMP-1

the diferences between groups A、B and groups C、D、E were significant(P<0.05).the diferences between groups C and E were not significant(P>0.05);To the expression of mRNA and protein of LMP-3

the diferences between groups A、B and groups C、D、E were significant(P<0.001).the diferences between groups D and E were significant(P<0.05);the expression of LMP-1 or LMP-3 between groups C and D were not significant(P>0.05).Conclusion:the constructed mammalian Co-expression plasmid pBudCE4.1-LMP-1-LMP-3 can express LMP-1 and LMP-3 molecules in vitro at the same time.