Objective To construct plant plasmid p2355-gtfB which contains multiple catalytic sites and antigen epitopes of glucosyltransferase of Streptococcus mutans. Methods Plasmid pcDNA3-gtfB was used as template to obtain the gtfB by PCR amplification. Then gtfB was inserted into inter-mediate vector pMD18-T, subcloned from the recombi-nant plasmid T-gtfB to plant expression vector p2355, then p2355-gtfB was transformed into Agrobacterium tumefaciens EHA105 by electroporation. Results Genetic segments 2. 9 kb and 3. 7 kb were obtained through restriction enzyme mapping analysis and DNA sequencing, and inserted gene 3 675 bp was shown in the construction recombinant plasmid p2355-gtfB through series analysis and detection. Conclusion The recombinant plasmid was constructed successfully. This research provided basis for further study on plants vaccine against dental caries.%目的 构建含变异链球菌葡糖基转移酶多个免疫显性抗原表位的植物表达质粒p2355-gtfB,为其转化植物研究奠定物质基础,为可食防龋疫苗研究提供条件.方法 以真核表达质粒pcDNA3-gtfB为模板,通过聚合酶链反应(polymerase chain reaction,PCR)扩增含编码变异链球菌葡糖基转移酶抗原表位的目的基因gtfB.将回收纯化的PCR产物经T-A克隆技术克隆于中间载体pMD18-T,双酶切鉴定插入方向后,将目的基因从中间载体释放,再克隆至高效的植物表达载体p2355,用电转化法转化根癌农杆菌EHA105,对重组质粒阳性克隆株进行筛选与鉴定.结果 通过对重组质粒T-gtfB进行酶切图谱分析,获得2.9kb和3.7 kb的2个片断,将重组质粒p2355-gtfB进行酶切、PCR及测序分析,显示p2355-gtfB中插入基因长度为3 675 bp,基因序列与双脱氧链终止法的测定结果相同,证明植物表达质粒p2355-gtfB构建成功,开放阅读框架正确.结论 本研究成功构建了含变异链球菌多个葡糖基转移酶免疫显性抗原表位编码基因的植物表达质粒p2355-gtfB,为可食性防龋疫苗的研究奠定了基础.
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