人骨形态发生蛋白2真核表达载体构建及其基因/壳聚糖纳米复合体制备  

Construction of human bone morphogenetic protein 2 and histidine eukaryotic expression plasmid and synthesis of chitosan/pIRES2-EGFP-hBMP2-His nanoparticles

作　　者：杨晓喻[1,2] 李世轶[1,2] 张迪[1,2] 吴颖[1,2] 杨涛[1,2] 刘长虹[1,2] 

机构地区：[1]南方医科大学附属口腔医院 [2]广东省口腔医院种植中心,广州510280

出　　处：《华西口腔医学杂志》2014年第5期493-497,共5页West China Journal of Stomatology

基　　金：国家自然科学基金资助项目(81170997)

摘　　要：目的构建人骨形态发生蛋白（BMP）2的真核表达型载体,并利用其与壳聚糖制备纳米复合体。方法体外利用双酶切法将pMD18T-hBMP2-His质粒与pIRES2-EGFP真核表达载体连接,构建pIRES2-EGFP-hBMP2-His真核表达载体;采用5种不同相对分子质量及脱乙酰度的壳聚糖,通过去溶剂法在不同N/P比（壳聚糖中的胺基含量与质粒中磷酸基含量的摩尔比）情况下制备壳聚糖/pIRES2-EGFP-hBMP2-His纳米复合体。ZetaPALS电位及粒度分析仪检测纳米复合体的粒径大小、分布及Zeta电位,琼脂糖凝胶电泳分析及荧光分光法评定壳聚糖对pIRES2-EGFP-hBMP2-His纳米复合体的包裹情况,原子力显微镜观察粒径形貌。结果 1）成功构建pIRES2-EGFP-hBMP2-His真核表达载体,并经酶切、测序证实;2）成功制备壳聚糖/pIRES2-EGFP-hBMP2-His纳米复合体,壳聚糖对pIRES2-EGFP-hBMP2-His质粒具有包裹作用;3）制备的壳聚糖/pIRES2-EGFP-hBMP2-His复合体呈球状,粒径为111.7-3 214.2 nm,Zeta电位为4.93-16.79 mV。结论壳聚糖可与pIRES2-EGFP-hBMP2-His复合形成纳米微粒。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 EcoRⅠ and 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 synthesized.

关 键 词：人骨形态发生蛋白2 真核表达载体 壳聚糖 纳米复合体 

分 类 号：R783.6[医药卫生—口腔医学]