霍乱毒素B亚单位植物细胞表达载体的构建及其在人参细胞中的表达

目的构建霍乱毒素B亚单位(CTB)植物细胞表达载体,并在人参细胞中进行表达。方法根据人参偏爱密码子,采用引物延伸PCR法合成CTB基因,连入pBI121质粒,构建植物细胞表达载体,转化人参细胞后,采用PCR、RT-PCR和Western blot进行鉴定。结果测序结果表明,PCR法合成的目的基因序列与设计完全一致,构建的植物细胞表达载体经双酶切鉴定显示,所含基因片段大小与预期相符。提取转基因人参细胞基因组DNA和mRNA,分别进行PCR和RT-PCR鉴定,均可见约400 bp的特异性片段。Western blot分析可见相对分子质量约12 000的特异性条带。结论已成功构建了含霍乱毒素B亚单位的植物细胞表达载体,并在人参细胞中获得表达。

Construction of Plant Expression Vector for Cholera Toxin B Subunit and Its Expression in Ginseng Cells

Objective To construct a plant expression vector for cholera toxin B subunit(CTB)and express in ginseng cells.Methods CTB gene was synthesized by primer extension PCR using the primers designed according to ginseng cell-preferred codonand inserted into plasmid pBI121.The constructed recombinant plasmid was transformed to ginseng cells,and the transformants wereidentified by PCR,RT-PCR and Western blot.Results The sequence of amplified target gene was completely identical to that de-signed.The restriction analysis of constructed plant expression vector proved that the length of inserted gene fragment was consistentwith that expected.Either PCR result of genomic DNA or RT-PCR result of mRNA of the transformed ginseng cells showed a specificgene fragment at length of about 400 bp.Western blot showed a specific band with relative molecular mass of about 12 000.Con-clusion A plant expression vector for CTB was successfully constructed and expressed in ginseng cells.