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 cryptococcous 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 -typehypersen tivityantigen 1,DHA1)与小鼠共刺激分子B 7-1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pUCmB 7-1TM和新型隐球菌重组质粒pcDNA3 -DHA1中特异扩增出编码B 7-1和DHA1的基因片段 ( 92 1bp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 该研究成功构建了新型隐球菌嵌合重组质粒pcDNA3 -B 7-1-DHA1。

Objective To construct the chimeric recombinant plasmid vector encoding costimulatory molecules B 7-1 and major cell immune response relevant gene DHA1 of cryptococcous neoformans. Methods Specific primers were designed to amplify the DNA fragments(PCR method) of B 7-1 and DHA1 genes from the recombinant plasmids pUCmB 7-1 TM and pcDNA3-DHA1 of cryptococcous neoformans respectively. The length of the acquired fragments were 921 bp and 984 bp. After being digested by appropriate restriction endoenzymes(HindⅢ,EcoRⅠand XbaⅠ), the generated fragments of B 7-1 and DHA1 genes were cloned into pcDNA3 one by one according to their specific orientations. Recombinant plasmid was identified by DNA sequencing and digestion 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-B 7-1 -DHA1 was successfully constructed in our study, which is a basic for our next study of cryptococcous neoformams DNA immunization.