幽门螺杆菌CagA蛋白与CTB融合基因的克隆及其植物表达载体的构建

The Cloning of the Fusion Gene of Helicobacter Pylori CagA and CTB and Construction of Its Plant Expression Vector

目的 构建能在水稻胚乳中特异表达的幽门螺杆菌细胞毒素相关蛋白(CagA)和霍乱毒素B亚单位(CTB)的植物表达载体.方法 采用高保真PCR技术分别从质粒pGEM CTB和幽门螺杆菌基因组DNA中扩增出CTB和cagA基因,然后用PCR方法直接融合CTB基因和cagA基因.又用PCR法改造了水稻Wx启动子,在其3′端融合加入Ω序列和Kozak序列.通过一系列亚克隆最终将两个融合基因和NOS终止子插入根癌农杆菌双元载体pCAMBI A1300的表达框架内.结果 PCR验证和测序分析证明,CTB和cagA融合基因以正确的方向插入到载体pCAMBI A1300中,并位于水稻Wx启动子后.结论 成功构建CTB和cagA融合基因的植物表达载体,为幽门螺杆菌口服疫苗的研究奠定基础.

ObjectiveThe aim of this research was to construct plant expression vector of Helicobacter pylori cytotoxin assiociated protein(CagA) and cholera toxin B subunit (CTB), which can be specifically expressed in rice albumen. Methods Cholera toxin B subunit gene(CTB) and Helicobacter pylori cytotoxin assiociated gene(cagA) were amplified by high-fidelity PCR respectively from plasmid pGEM-CTB and Helicobacter pylori(H.pylori) genomic, and then they were fused by PCR.The Wx promoter of rice was modified by PCR, and Ω sequence and Kozak sequence were fused into 3′ terminal of the Wx promoter. After several steps of sub-cloning, both fusion genes and NOS terminator were finally inserted into the expression frames of binary vector pCAMBIA1300 of Agrobacterium tumefacieris. ResultsPCR and DNA sequencing confirmed that the fusion gene of cagA and CTB had been correctly inserted into vector pCAMBIA1300,and was placed behide the Wx promoter of rice. ConclusionsThe plant expression vector of the fusion gene of cagA and CTB has been constructed successfully, which laid the foundation for developing an oral vaccine against H.pylori infection.