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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 pS BR CTA2 B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 CTA2 B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 pMD1 8 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 pcD NA3 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 immunostimulatory sequence capable of preventing Streptococcus mutans from adhering to the tooth surface. Methods: According to the sequence of prokaryotic expression plasmid pS BR CTA2 B, a specific PCR primer targeting to the sialoprotein-binding segment (SBR) in Ag Ⅰ / Ⅱ of Streptococcus mutans surface antigen was designed and amplified by PCR from plasmid pSBR CTA2 B The target DNA fragment was cloned into the intermediate vector pMD18T by TA cloning technique. After identifying the insertion direction, the clones with the correct orientation were inserted and the target DNA was released from the intermediate vector and cloned into the vector containing the CpG immunostimulatory sequence Eukaryotic expression vector pcDNA3 1. Results: The recombinant plasmid pcDNA3 1 SBR was digested with restriction endonuclease analysis and DNA sequence analysis. The result showed that the eukaryotic recombinant plasmid pcD NA3 1 SBR was successfully constructed and the open reading frame was correct. Conclusion: The DNA fragment encoding SBR can be successfully cloned into the appropriate site of the eukaryotic expression vector pcDNA3 1 containing the immunostimulatory sequence CpG by TA cloning technology, and the eukaryotic recombinant plasmid pcDNA 3 1 SBR can be constructed as an effective anticaries DNA vaccine