Construction of recombinant gene adenovirus containing human LIM mineralization protein-1 and its expression in bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: LIM Mineralization protein 1 (LMP-1), an intracellular non-secretory protein, plays roles in bone calcification. Presently, it is found that LMP-1 can promote an increase in bone morphogenetic protein 2 and transforming growth factor ? 1. This indicates that LMP-1 may recruit a mass of ossified factors to participate in the differentiation of osteoblasts. OBJECTIVE: To construct human LMP-1 gene adenovirus recombinant with AdEasy adenovirus vector system, and to detect LMP-1 expression in infected rabbit bone marrow mesenchymal stem cells (BMSCs). DESIGN, TIME AND SETTING: An opening experiment was performed at the Central Laboratory of South West Hospital, Third Military Medical University of Chinese PLA from March 2006 to February 2007. MATERIALS Three New Zealand rabbits were used to isolate bone marrow mesenchymal stem cells. Plasmid pIRES2-EGFP-LMP-1 carrying human LMP-1 gene was kept in Department of Orthopedics, Second Hospital Affiliated to Chongqing Medical University. AdEasy was presented by Dr. Tong-Chuan He from USA. Human embryo kidney 293 cells were gifted by Wang from Department of Clinical Laboratory of Chongqing Medical University. METHODS: LMP-1 gene with a sequence encoding His-tag was amplified by using pIRES2-EGFP-LMP-1 plasmid as a template for polymerase chain reaction (PCR) with a specially designed downstream primer. The target gene was cloned to the pMD18-T vector for sequencing. Once verified, the gene was cut out by double endonucleases, connected to the shuttle vector pAdTrack-CMV. The newly constructed vector was linearized by PmeⅠ following efficient homologous recombination with the backbone vector pAdEasy-1 in BJ5183. The correct recombinant pAd-LMP-1 was linearized with Pac I and transfected to HEK293 cell by means of mediated Lipofectamine. The titer of virus was measured after amplification and purification. The mRNA and protein expression of LMP-1 was detected in BMSCs, which were infected with Ad-LMP-1 at the most appropriate MOI, were detected by reverse transcription (RT)-PCR and Western blot, respectively. MAIN OUTCOME MEASURES: Plasmid pAd-LMP-1 identification, its titre and efficiency of infection. mRNA and protein expression of LMP-1 were detected by RT-PCR and Western blot, respectively. RESULTS: The recombinant plasmid pMD18-T-LMP-1 carrying LMP-1 gene with His-tag was successfully constructed. After packaging and amplification of the recombinant adenovirus, the 3.5?109 efu/ml titer of Ad-LMP-1 was obtained by CsCl gradient purification. The optimal efficiency infection was 50%-70%, which was get after Ad-LMP-1 infected BMSCs for 3 days at the most appropriate MOI 150. The mRNA and protein expression of LMP-1 in infected BMSCs had been proved. CONCLUSION: The recombinant adenovirus containing human LMP-1 gene with His-tag is successfully constructed. The BMSCs infected with recombinant adenovirus Ad-LMP-1 can effectively express LMP-1.