Construction of human bone morphogenetic protein 2 and histidine eukaryotic expression plasmid and synthesis of ;chitosan/pIRES2-EGFP-hBMP2-His nanoparticles
- VernacularTitle:人骨形态发生蛋白2真核表达载体构建及其基因/
- Author:
Xiaoyu YANG
1
;
Shiyi LI
;
Di ZHANG
;
Ying WU
;
Tao YANG
;
Changhong LIU
Author Information
- Keywords: human bone morphogenetic protein 2; eukaryotic expression vector; chitosan; nanoparticle
- From: West China Journal of Stomatology 2014;(5):493-497
- CountryChina
- Language:Chinese
- Abstract: Objective To clone and construct a eukaryotic expression vector of human bone morphogenetic protein (BMP) 2 and histidine in vitro and synthesize chitosan (CS)/pIRES2-EGFP-hBMP2-His nanoparticles. Methods pMD18T-hBMP2-His was digested by EcoRⅠand BamHⅠto obtain the hBMP2-His gene, which was inserted into pIRES2-EGFP to form pIRES2-EGFP-hBMP2-His. Afterward, CS, which exhibited five different molecular weights and deacetylation degrees, was complexed with pIRES2-EGFP-hBMP2-His to form CS/pIRES2-EGFP-hBMP2-His nanoparticles; in this procedure, a desolvent method was used at different N/P ratios (amino in CS to phospho in plasmid DNA). The gene-encapsulating ability of CS was evaluated by agarose gel electrophoresis and fluorescence spectrophotometry; size, distribution, and potential were analyzed using a ZetaPALS analyzer. The shape of the nanoparticles was observed under an atomic force microscope. Results 1) pIRES2-EGFP-hBMP2-His was constructed after the cloned hBMP2-His gene was confirmed by sequencing. 2) CS/pIRES2-EGFP-hBMP2-His nanoparticles were synthesized and pIRES2-EGFP-hBMP2-His was packaged by CS. 3) CS/pIRES2-EGFP-hBMP2-His nanoparticles were globular with an average size of 111.7 nm to 3 214.2 nm and an average zeta-potential of 4.93 mV to 16.79 mV. Conclusion CS/pIRES2-EGFP-hBMP2-His nanospheres are successfully syn-thesized.
