Journal of Jilin University Medicine Edition ›› 2015, Vol. 41 ›› Issue (01): 32-38.doi: 10.13481/j.1671-587x.20150107

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Construction of recombinant adenovirus of mouse MafB gene and its effect on osteoclast differentiation

ZHU Yun1, WANG Zhengyu1, YANG Xiaozhong1, MA Tao1, CHEN Tingmei2, ZHANG Jian1   

  1. 1. Department of Orthopaedics, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China;
    2. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China
  • Received:2014-06-04 Published:2015-01-30

Abstract:

Objective To construct the recombinant adenovirus vector of mouse MafB gene, and to elucidate the influence of MafB in osteoclast differentiation. Methods The total RNA of RAW264.7 cells in mouse was extracted and reversedly transcripted to cDNA, and the target gene MafB was obtained by RT-PCR with cDNA as template.The MafB gene was connected to shuttle vector pAdTrack-CMV and linearized with PmeⅠ and transfected to competent E.coli BJ5183 containing pADEasy-1.The adenovirus Ad-MafB in HEK 293 cells was amplified after linearization with Pac Ⅰ and the RAW264.7 cells were infected.The RAW264.7 cells were devided into control group, empty-vector group(Ad-GFP group) and over-expression group(Ad-MafB group).The expression levels of MafB were detected by RT-PCR and Western blotting method.TRAP staining was applied to observe the osteoclast differentiation and maturation.RT-PCR and Western blotting method were applied to detect the expression of TRAP and cathepsin K(CTSK). Results Compared with control group and Ad-GFP group, the expression levels of MafB mRNA and protein in Ad-MafB group were increased(P<0.05).The TRAP staining results showed that the number of TRAP-positive cells in Ad-MafB group was less than those in control group and Ad-GFP group.Compared with control group and Ad-GFP group, the expression levels of TRAP and CTSK mRNA and proteins in Ad-MafB group were decreased(P<0.05). Conclusion The recombinant adenovirus Ad-MafB over expressing MafB in mouse RAW264.7 cells is successfully constructed, and it can inhibit the differentiation of osteoclasts.

Key words: v-maf musculoaponeurotic fibrosarcoma oncogene family,protein B, osteoclast, recombinant adenovirus, cell differentiation

CLC Number: 

  • Q782