摘要
目的 :采用基因重组技术 ,将结核杆菌Ag85A与协同刺激信号CD80基因融合 ,构建成融合结核DNA疫苗 ,旨在提高结核DNA疫苗的免疫效果 ,可望通过基因免疫 ,获得免疫原性强 ,以低剂量即可有效刺激机体产生T细胞和B细胞应答 ,安全可靠的新型结核病疫苗 .方法①pcDNA3-tPA -Ag85A质粒的构建和鉴定以V1Jns-tPA-Ag85A质粒为模板 ,PCR扩增tPA -Ag85A ,(不含终止密码 ) ,定向插入pcDNA3载体的BamHⅠ和EcoRⅠ位点之间 ,构建成pcDNA3-tPA -Ag85A质粒。②pcDNA3-tPA -Ag85A/CD80质粒的构建与鉴定以pcDNA3CD80质粒为模板 ,扩增CD80 (含终止密码 )定向插入pcDNA3-tPA -Ag85A质粒的EcoRⅠ和XbaⅠ位点之间 ,构建成pcDNA -tPA-Ag85A/CD80融合DNA真核表达质粒。结果 :重组质粒pcDNA3-tPA -Ag85A和pcDNA3-tPA -Ag85A/CD80经酶切鉴定和测序鉴定均构建正确。结论 :结核杆菌Ag85A与协同刺激分子CD80融合DNA真核表达质粒构建成功 ,为进一步研究比较其免疫保护效果 。
Objective: To construct and identify the chimeric Ag85A/B7 eukaryotic expression plasmid.Methods:A.Construction of an eukaryotic expression plasmid pcDNA3-tPA85A The full length of Ag85A gene was kindly donated by Professor. Kris Huygen DNA fragment with tPA signal sequence(without stop codon) was amplified by PCR with BamHⅠ and EcoRⅠ site containing primers. Amplified DNA was digested with BamHⅠ and EcoRⅠ, then was subcloned into the unique BamHI and EcoRI cloning sites of pcDNA3 expression vector B. Construction of an eukaryotic expression plasmid pcDNA3-tPA85A/CD80: CD80 fragment (including stop codon) was amplified by PCR using primers designed to generate EcoRⅠ and XbaⅠ restriction sites at the 5'and 3'ends of the amplified fragments, respectively. Then the amplified CD80 DNA was subcloned into unique EcoRⅠ and XbaⅠ cloning sites of the plasmid pcDNA3-tPA-85A.Thus,the N terminus of CD80 gene was fused to the C terminus of tPA-85A gene. The constructed pcDNA3-tPA-85A and pcDNA3-tPA-85A/CD80 plasmid were identified. Results:Recombinant plasmids pcDNA3-tPA-85A and pcDNA3-tPA-85A/CD80 were constructed and their accuracy was confirmed by restriction enzyme digestion and DNA sequencing.Conclusions:Recombinant plasmids pcDNA3-tPA-85A and pcDNA3-tPA-85A/CD80 were constructed correctly. The construction of the chimeric Mycobacterial tuberculosis Antigen85A/costimulatory molecule CD80 Eukaryotic Expression plasmid provided the possibility for studying the new, safe, and effective tuberculosis vaccine.
出处
《泸州医学院学报》
2003年第5期382-385,共4页
Journal of Luzhou Medical College
