B7-1和新型隐球菌DHA1基因嵌合重组质粒的构建

Construction of the Chimeric Recombinant Plasmid vector Encoding Costimulatury molecules mB7- 1 and DHA1 Gene Of Cryptococous Neoformans

目的构建包含新型隐球菌细胞免疫主要相关基因—迟发超敏反应抗原基因(delayed-type hypersentivity antigen 1,DHA1)与小鼠共刺激分子B7-1的嵌合重组质粒。方法设计合成两对寡核苷酸引物,用PCR法分别从pUCmB7-1TM和新型隐球菌重组质粒pcDNA3-DHA1中特异扩增出编码B7-1和DHA1的基因片段(921和984bp),分别用HindⅢ、EcoRⅠ、XbaⅠ酶切后,逐个定向连接到质粒pcDNA3中,转化宿主菌DH-5α,分别用上述内切酶酶切及DNA测序鉴定重组质粒。结果酶切鉴定示所切下的片段大小均与预计相符,测序结果与文献报道序列及预计结果一致,证实符合表达框架。结论本实验成功构建了新型隐球菌嵌合重组质粒pcDNA3-B7-1-DHA1,为进一步研究新型隐球菌DNA免疫奠定了基础。

Objective To construct the chimeric recombinant plasmid vector encoding costimulatory molecules B7-1 and major cell immune response relevant gene DHA1 of Cryptococcous neoformans. Methods The specific primers were designed synthesized to amplify the DNA fragments (PCR method) of B7-1 and DHA1genes from the recombinant plasmids pUCmB7-1TM and pcDNA3-DHA1 of Cryptococcous neoformans respectively. The length of the acquired fragments were 921bp and 984bp. After being digested by appropriate restriction endoenzymes (HindⅢ, EcoRⅠ and XbaⅠ) , the generated fragments of B7-1 and DHA1 genes were cloned into pcDNA3 one by one according to their specific orientations. Then the recombinant plasmid was identified by DNA sequencing and digesting of endoenzymes. Results The fragments digested by endoenzymes were as large as the predicted results. DNA sequencing was the same with the sequence reported on the literatures. Conclusion The chimeric recombinant plasmid pcDNA3-B7-1-DHA1 was successfully constructed in our study. It is a basic for our next study of cryptococcous neoformams DNA immunization.