广范围高效的植物乙型肝炎病毒表面抗原表达载体构建及鉴定

目的构建含乙型肝炎病毒表面抗原（HBsAg）基因的植物双元表达载体。方法采用高保真PCR方法从T—adr质粒扩出HBsAg基因，定向克隆到中间载体pUC18-DHA，经测序证实核酸序列正确后，再亚克隆到植物双元表达载体pGreen0029-GFP上，采用电击法将含HBsAg的植物表达载体转入根癌农杆菌中，并进行酶切证实。结果扩增的HBsAg片断亚克隆到中间载体，得到pUC18-HBsAg—DHA，测序证实HBsAg核酸序列正确，再与根癌农杆菌双元载体pGreen0029-GFP连接，获得含HBsAg基因的植物双元表达载体pGreen0029-HBsAg—DHA，转入根癌农杆菌，酶切证实正确。结论采用正确技术路线，构建了含HBsAg基因的植物表达载体，为下一步的转基因植物表达研究奠定了基础。

Construction and identification of HBV surface antigen wide plant species and high efficiency expression vector

Objective: To construct the high efficiency plant expression vector containing HBV surface antigen gene(HBsAg).Methods Using high-fidelity PCR,HBsAg was amplified from T-adr plasmid and then subcloned into the transition vector pUC18-DHA.Following the confirmation of the HBsAg sequence,the vector was subcloned into the plant expression vector pGreen0029-GFP that was subsequently transferred into the Agrobacterium tumefaciens G3101 by electroporation.Results the HBsAg DNA that was ligated into the transition vector pUC18-DHA resulted in the pUC18-HBsAg-DHA.After the vector was ligated into the plant binary vector pGreen0029-GFP,new plant binary vector,pGreen0029-HBsAg-DHA,was produced.Analysis with restriction endonucleases confirmed successful transfer of pGreen0029-HBsAg-DHA into Agrobacterium tumefaciens G3101.Conclusion With appropriate technological strategy,the plant binary expression vector encoding HBsAg has been constructed,which facilitates further investigation of HBsAg protein expressions in transgenetic plant.