通过T-A克隆技术构建含免疫刺激序列CpG基序的抗SBR DNA疫苗

摘要

目的:构建含免疫刺激序列、能够阻止变形链球菌在牙面粘附的防龋DNA疫苗.方法:根据原核表达质粒pSBR-CTA2B基因序列,设计针对编码变形链球菌表面蛋白抗原AgⅠ/Ⅱ分子中唾液蛋白结合区段(SBR)的特异性PCR引物,利用PCR技术由质粒pSBR-CTA2B扩增目的DNA片断;通过T-A克隆技术将目的DNA片段克隆于中间载体pMD18-T,鉴定插入方向后,选择插入方向正确的克隆,将目的DNA从中间载体释放,再克隆至含有CpG免疫刺激序列的真核表达载体pcDNA3.1.结果:通过对重组质粒pcDNA3.1-SBR进行酶切图谱分析和DNA序列测定分析,证明真核表达重组质粒pcDNA3.1-SBR构建成功、开放阅读框架正确.结论:利用T-A克隆等技术可成功将编码SBR的DNA片段克隆到含有免疫刺激序列CpG的真核表达载体pcDNA3.1的适当部位,构建出真核表达重组质粒pcDNA 3.1-SBR作为有效防龋的DNA疫苗.%Objective:To construct an anti-caries DNA vaccine containing immumostimulatory oligonucleotide CpG motif for preventing Streptococcus mutans from binding to the tooth furfaces.Methods:According to the sequence of the plasmid pSBR-CTA2B,a pair of primers specific for amplifying the DNA fragment encoding saliva-binding region(SBR)in surface protien AntigenⅠ/Ⅱ(AgⅠ/Ⅱ)molecule from S.mutans were designed and synthesized.With PCR,the target DNA fragment encoding SBR was amplified from plasmid pSBR-CTA2B.Through T-A cloning,the target DNA fragment was first inserted into plasmid intermediate vector pMD18-T,and then subcloned from the recombinant plasmid pMD 18-T-SBR to the eukaryotic expression vector pcDNA 3.1 into which the immunostimulatory oligonucleotide CpG motif had been incorporated.Results:Through restriction enzyme mapping analysis and DNA sequencing,the construction of the eukaryotic expression recombinant plasmid pcDNA 3.1-SBR with expected open reading frame was confirmed.Conclusion:By T-A cloning,the DNA fragment encoding SBR can be cloned to vector pcDNA 3.1 bearing immunostimulatory sequence to construct the recombinant plasmid pcDNA 3.1-SBR possissing the property of immunogenic enhancement,and the recombinant plasmid can be further used as a novel and more effective anti-caries vaccine.