含变形链球菌PAcA基因的真核表达质粒pcDNA3.1-PAcA的构建与鉴定  

Construction and characterization of a eukaryotic expression plasmid pcDNA3.1-PAcA containning streptococcus mutans PAcA

作　　者：杨小青[1] 姜广水[2] 张兆莲[1] 王晶 杨丕山[1] 刘贤锡[2] 卞继峰[2] 胡海燕[2] 耿昭[2] 卢翌[2] 

机构地区：[1]山东大学口腔医学院 [2]山东大学医学院分子生物学实验中心 [3]枣庄市市中区医院

出　　处：《山东大学学报（医学版）》2002年第4期289-291,共3页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题 ( 30 1710 10 );山东医科大学重点课题启动基金资助课题 ( 4130 2 6 )

摘　　要：目的 :构建含有免疫刺激序列、可防止变形链球菌在牙面粘附的DNA疫苗。方法 :利用PCR技术由质粒pPAcA CTA2 B扩增编码PAcA的DNA片段 ;通过T A克隆技术将目的DNA片段克隆于载体pMD1 8 T ,鉴定插入方向后 ,将目的DNA从载体上释放 ,再克隆于含有CpG免疫刺激序列的真核表达载体pcDNA3 .1 ,构建出用作防龋疫苗的真核表达质粒pcDNA3 .1 PAcA。结果 :通过对重组质粒pcDNA3 .1 PAcA进行酶切图谱和DNA序列测定分析 ,证明真核表达重组质粒pcDNA3 .1 PAcA构建成功、阅读框架正确。结论 :利用T A克隆等技术可成功将编码PAcA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 ,构建出真核表达重组质粒pcDNA3 .1 PAcA。Objective: To construct an anti caries genetic vaccine containing immunostimulatory oligonucleotide CpG motif for preventing Streptococcus mutans from binding to the tooth surfaces. Methods: With PCR,the target DNA fragment encoding PAcA of Streptococcus mutans was amplified from plasmid pPAcA CTA 2B.Through T A cloning,the target DNA fragment was first inserted into plasmid vector pMD18 T,and then subcloned from the recombinant plasmid pMD18 T PAcA with expected open reading frame to the eukaryotic expression vector pcDNA3.1 into which the immunostimulatory oligonucleotide CpG motif had been incorporated. Results: The construction of the eukaryotic expression recombinant plasmid pcDNA3.1 PAcA with expected open reading frame was confirmed through restriction enzyme mapping analysis and DNA sequencing. Conclusion: By T A cloning,the DNA fragment encoding PAcA can be cloned to vector pcDNA3.1 bearing immunostimulatory sequence to construct the recombinant plasmid pcDNA3.1 PAcA possessing the property of immunogenic enhancement.

关 键 词：龋齿 疫苗构建 CPG基元 T-A克隆 变形链球菌 

分 类 号：R781.1[医药卫生—口腔医学]