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Construction and package of pAAV-hBMP2-IRES plasmid

CHENG Zheng,WANG Min,SHI Jian-feng,SHI Zhi-bin.Stomatological Hospital of Xi'an Jiaotong University,Xi'an,710004,China.  
Objective:To construct the recombinant adeno-associated virus vector with human bone morphonenetic protein 2 gene(rAAV-hBMP2).Method:The moulds which contained hBMP-2 vector were amplified by PCR.The re-claimed productions and pAAV-IRES-GFP were digested and integrated,bacillus coli DH5α competent cells were trans-formed,and recombinant plasmid pAAV-hBMP2-IRES-GFP was obtained.293 cells were virus-encloded by the calcium phosphate precipitation method and the virus fluid was obtained and processed by chloroform precipitate treatment,PEG8000/NaCI precipitation and chloroform extraction for purification.Virus titer was identified by fluorescence counting method.Result:Calcium phosphate coprecipitation methods were used in 293 cells and adeno-associated viral vector rAAV-IRES-hBMP2 was recombinated.The virus titer was about 3.6×1011/ml.Conclusion:With this method,rAAV-hBMP2 can be acquired successfully and it is useful to bone tissue engineering.
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