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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 the plant expression vector of Streptococcus mutans surface protein PAc A and cholera toxin B subunit and provide the experimental basis for the further study of the edible chimeric dental caries vaccine. Methods The fragment containing 35s promoter, NOS terminator and multiple cloning site was obtained from the intermediate vector pBPC55 and ligated into the plant binary expression vector pCAMBIA2301 to construct the recombinant vector p2355. The surface protein was obtained from the recombinant plasmid pEAC10 by PCR amplification The fusion gene pacA-ctxB between PAcA region and cholera toxin B subunit was cloned into recombinant plasmid p2355 to construct plant expression vector p2355-PAcA / CTB, and electrotransformation into Agrobacterium tumefaciens EHA105. PCR and restriction enzyme digestion. Results The 1.1kb fragment containing 35s promoter, NOS terminator and multiple cloning site was amplified by PCR. The fragment was inserted into the plant binary vector pCAMBIA2301. The gene of pacA-ctxB was amplified by PCR. The constructed plasmid p2355-PAcA / CTB was digested with XhoI and KpnI and detected by PCR. The 1.7kb fragment was obtained, which was the same size as the predicted fragment. Conclusion The plant expression vector p2355-PAcA / CTB with pacA-ctxB fusion gene was successfully constructed.