Construction Of Co-expression Plasmid Pbudce4.1-lmp-1-lmp-3 And Its Expression In Vitro

Objective:With the increasing development of Genetic engineering and tissue engineering technique, it provides more therapeutic selections for clinical bone defects and lumbar infusion. Nowadays, LIM mineralization protein (LMP)has been a hot issue. This experiment is to construct the mammalian CO-expression plasmid pBudCE4.1-LMP-1- LMP-3,plasmid pBudCE4.1-LMP-1 and plasmid pBudCE4.1-LMP-3,which were transfected into MSCs respectively. the expression of LMP-1 and LMP-3 were detected in each group so as to compare double gene group with single gene group respectively. Theoritcal supports were provided to do animal experiments by this experiment.Methods:1,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. 2,fragments of LMP-1 and LMP-3 gene were constructed respectively into the plasmid vector pBudCE4.1,fragments of LMP-3 gene were constructed into the plasmid vector pBudCE4.1-LMP-1 after identification with nucleotide sequencing and enzymes. 3,rabbit MSCs were isolated and cultured,the cells were identified by the microscope. MSCs cell line was transfected with this Co-expression plasmid using lipofectin reagent. according to the transfected 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). 4,the expression of LMP-1 and LMP-3 were detected by RT-PCR and Western blot technique,the expression differences were analysed at the end.Results: 1,The plasmids Puc57-LMP-1,Puc57-LMP-3,pBudCE4.1- LMP-1,pBudCE4.1-LMP-3 and pBudCE4.1-LMP-1- LMP-3 were obtained successfully and verified by nucleotide sequencing and enzymes. 2,After transfection with this mammalian Co-expression plasmid,the LMP-1 and LMP-3 molecules were expressed in MSCs cells. 3,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: 1,The plasmids pBudCE4.1-LMP-1,pBudCE4.1 -LMP-3 and pBudCE4.1-LMP-1-LMP-3 were obtained successfully 2,After transfection with this mammalian Co-expression plasmid,the LMP-1 and LMP-3 molecules were expressed in MSCs cells. 3,the expression level of LMP-3 was improved in group E in comparison with group D or group C,reciprocal expression among LMP-1and LMP-3 was testified.