乙肝病毒表面抗原基因在胡萝卜中的克隆及表达

构建HBV表达抗原（HBsAg)植物表达载体并在胡萝卜植株中表达。采用自行构建adr亚型HBsAg基因克隆T-adr，再次酶切获得860bp含PreS2的HBsAg基因片段，将其插入到植物表达载体pBPC55，新质粒命名为pBPC91adr。将其与含除草剂抗性基因及GUS蛋白基因的筛选质粒pBPC93共同经基因枪（PDS-1000/He)转化胡萝卜悬浮细胞，经含除草剂（Biolaphos)的培养基筛选及植物激素诱导分化，获得除草剂抗性胡萝卜幼苗，结果为转化后8周，自胡萝卜细胞中分化出除草剂抗性胡萝卜幼苗，提取新分化幼苗总DNA，特异性引物PCR扩增后可见860bp扩增带；Southern-Blot证明有HBsAg基因整合，胡萝卜蛋白萃取物的Western-Blot及ELISA检测证实有HBsAg蛋白表达。利用基因枪转化使质粒pBPC91adr中HBsAg基因在胡萝卜幼苗内整合并表达，提示以植物生产疫苗具有可行性。

Cloning and expression of the hepatitis B surface antigen in carrots

In order to establish a plant expressing system which could express HBsAg in carrots.The 860 bp HBsAg gene of adr subtype was cut by restriction enzyme from plasmid T adr and inserted into pBPC55.The new plasmid was named as pBPC91adr.It was cotransformed with selecting plasmid pBPC93 which offers GUS and anti Biolaphos gene into carrot cells via Microprojectile system.There were carrot shoots differentiated from anti Biolaphos cells after 8 weeks selection, the shoots were tested with following methods:Total DNA extraction from the anti Biolaphos could be amplified a 860 bp segment by PCR,and Southern Blot showed that HBsAg gene was successfully transformed;Western Blot and ELISA tests showed that the HBsAg protein had been produced in anti Biolaphos carrots.The HBsAg gene in constructed plasmid pBPC91adr has been successfully expressed in carrots,this study proves that transgenic plant could be used as a candidate vector to produce vaccine or orther protein of medical improtance.