Construction and identification of human pIRES-hBMP–2 eukaryotic expression recombinant

Objectives: To construct eukaryotic expression recombinant plasmid of human bone morphongenetic protein-2. Methods: The result of pMD18-T-BMP-2 insert fragment sequence determination and the atlas of plasmid vector pIRES were refered, a pair of primers was designed which contained two restriction sites: EcoR±and Sal±.Then BMP-2 cDNA fragment were amplified from prokaryotic expression recombinant pMD18-T-BMP-2 by polymerase chain reaction. The purified PCR product and empty plasmid pIRES were digested by EcoR±and Sal±, the PCR product and empty plasmid were linked by T4 link enzyme in the proportion of 2:1. competence E.coli DH5a was Prepared. Then the link product were transformed to competence E.coli DH5a. The single cloned bacillus selected. The E.coli DH5a were cultured in 37°C for 12-16 hours .Then 1-4 millilitre E.coli DH5a was taken and plasmid was extracted from E.coli DH5a. Digested by restriction endonuclease, the product was identified through Agarose electrophoresis and observed if pIRES-rhBMP-2 were present in the E.coli DH5a liquid. Sequence analysis of positive recombinant was carried out by automatic DNA Sequencer. Results: 1.PCR amplified hBMP-2 cDNA fragment. A 1196 base pair single DNA fragment was found in the PCR products by 20g/L agarose electrophoresis, the same size as expected for BMP-2 cDNA. However not any special band was found in negtive contrast.2. The construct of recombinant: The PCR product and plasmid vector was digested by EcoR±and Sal±separately, transformed competence E.coli DH5a ,There were many bacteria in the empty plasmid transformed culture flat ,while there was not any bacterium existing in the negative contrast culture flat.3.The sieving of positive recombinant and sequence determine. The insertion fragment could be seen after restriction digestion as expected. Sequence analysis showed the same sequence as expected. Conclusions: PIRES-hBMP-2 eukaryotic recombinant has been constructed successfully,which provides a sound basis for further research of periodontal disease.