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 EcoR I and BamH I to 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.
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。