植物细胞表达人胰岛素的载体构建

Construction of Vector for Expression of Human Insulin in Plant Cells

目的构建人胰岛素的植物细胞表达载体。方法设计多对引物,通过PCR反应合成霍乱毒素B亚单位(CTB)基因和不带C肽的人胰岛素基因。将霍乱毒素B亚单位(CTB)基因与这种不带C肽的人胰岛素基因融合后,插入克隆载体,然后用BamHⅠ和SacⅠ进行酶切,并将酶切的融合基因片段连接在植物表达载体pBI121上,最后用BamHⅠ和SacⅠ进行酶切鉴定。结果测序结果表明,PCR扩增的目的基因顺序与设计完全一致,并构建了克隆载体和植物表达载体。植物表达载体经酶切后鉴定,所含基因片段大小与预期相符。结论已成功地构建了人胰岛素基因的植物细胞表达载体。

Objective To construct the vector for expression of human insulin in plant cells. Methods The cholera toxin B subunit（ CTB ） gene and human insulin gene without C peptide were synthesized by PCR using the designed primers ,then fused and inserted into cloning vector pMD18-T. Digest the recombinant plasmid with BamH Ⅰand Sac Ⅰ ,and clone the obtained fusion gene into expression vector pBI121. Identify the recombinant plasmid by digestion with BamH Ⅰ and Sac Ⅰ . Results The sequences of synthetic CTB and human insulin genes were completely identical to those designed. Restriction map proved that the length of gene fragment inserted into expression vector pBI121 was consistent with that expected. Conclusion The vector for expression of human insulin in plant cells was successfully constructed.