变异链球菌表面蛋白PAc基因A区与霍乱毒素B亚单位融合基因植物表达载体的构建

Constructing plant expression vector carrying Fusion gene of A region of PAc of Streptococcus mutans and cholera toxin B subunit

目的构建变异链球菌表面蛋白PAc A区与霍乱毒素B亚单位的植物表达载体,为可食嵌合体防龋疫苗的进一步研究提供实验基础。方法从中间载体pBPC55获得含35s启动子、NOS终止子和多克隆位点的片段,定向连接到植物双元表达载体pCAMBIA2301上,构建重组载体p2355;通过PCR扩增从重组质粒pEAC10中获得表面蛋白PAcA区与霍乱毒素B亚单位的融合基因pacA-ctxB,克隆至重组质粒p2355中,构建植物表达载体p2355-PAcA/CTB,电转化法导入根癌农杆菌EHA105。PCR及酶切鉴定。结果 PCR扩增得到了含35s启动子、NOS终止子和多克隆位点的1.1kb大小片段,经酶切鉴定,该片段已经插入到植物双元载体pCAMBIA2301中。PCR扩增得到了pacA-ctxB基因;构建的质粒p2355-PAcA/CTB经XhoⅠ和KpnⅠ双酶切及PCR检测,均得到1.7kb大小的片段,与预计的嵌合目的基因片段大小相同。结论成功构建pacA-ctxB融合基因的植物表达载体p2355-PAcA/CTB。

Objective To construct plant expression vector carrying fusion gene of A region of PAc Streptococcus mutans and cholera toxin B subunit so as to provide useful information for further study on edible vaccine against dental caries.Methods The recombination vector p2355 was constructed using pCAMBIA2301 which was insterted with 35s promoter,MCS and nos terminus from pBPC55.With PCR,the target DNA fragment encoding A region of PAc of Streptococcus mutans and B subunit of cholera toxin was obtained and inserted to p2355.So the highly-expressed vector p2355-PAcA/CTB was constructed,and introduced into an Agrobacterium strain,EHA105.The plant expression vector was identified by PCR and restriction enzyme digestion.Results Restriction endonuclease comfirmed that plant binary vector p2355 was constructed successfully.The 1.4kb fused gene pacA-ctxB of the recombination plasmid p2355-PAcA/CTB were obtained by restriction endonuclease digestion and PCR.Conclusions Successfully constructed transgenic plant expression vector p2355-PAcA/CTB harbying pacA-ctxB.