| 白假丝酵母菌天冬氨酸蛋白酶真核表达质粒pcDNA3．1／SAP2的构建 |
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| 引用本文： | 李蕾,邹先彪,杨宇光. 白假丝酵母菌天冬氨酸蛋白酶真核表达质粒pcDNA3．1／SAP2的构建[J]. 中国感染控制杂志, 2008, 7(4): 233-236 |
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| 作者姓名： | 李蕾 邹先彪 杨宇光 |
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| 作者单位： | 解放军总医院第一附属医院,北京,100037 |
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| 摘 要： | 目的构建白假丝酵母菌天冬氨酸蛋白酶真核表达质粒pcDNA3．1／SAP2．为以其作为基因疫苗进行免疫动物实验奠定物质基础。方法从白假丝酵母菌中提取基因组DNA，以其为模板采用聚合酶链反应（PCR）法获取SAP2基因，将真核表达质粒pcDNA3．1（＋）myc-HisC和SAP2基因行EcoRI和XhoI双酶切，琼脂糖凝胶纯化，连接酶切产物，转化TOP10感受态细菌，筛选菌落和测序鉴定。结果经PCR扩增获得的目的基因分子量与预计相同，并定向插入真核表达载体pcDNA3．1（＋）myc-HisC，电泳获得预期的SAP2条带,测序证实为正确的SAP2序列。结论利用真核表达质粒pcDNA3．1（＋）myc-HisC可以方便而高效地构建pcDNA3．1／SAP2重组质粒，该质粒能直接激活抗原提呈细胞，可以使重组质粒具有较强的免疫原性和免疫反应性。
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| 关 键 词： | 白假丝酵母菌 天冬氨酸蛋白酶 SAP2基因 重组质粒 基因疫苗 |
Construction of eukaryotic expression plasmid pcDNA3.1/SAP2 expressing secretive aspartyl proteinase2 of Candida albicans |
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| LI Lei,ZOU Xian-biao,YANG Yu-guang. Construction of eukaryotic expression plasmid pcDNA3.1/SAP2 expressing secretive aspartyl proteinase2 of Candida albicans[J]. Chinese Journal of Infection Control, 2008, 7(4): 233-236 |
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| Authors: | LI Lei ZOU Xian-biao YANG Yu-guang |
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| Affiliation: | (The First Affiliated Hospital of People＇s Liberation Army General Hospital, Beijing 100037, China) |
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| Abstract: | Objective To construct eukaryotic expression plasmid pcDNA3. 1/SAP2 expressing secretive aspartyl proteinase2 （SAP2） of Candida albicans, so as to lay a foundation for animal immunization test based on this gene vaccine. Methods The target gene fragment SAP2 was obtained by standard PCR amplification, then SAP2 and plasmid pcDNA3.1 （＋）myc-HisC were cleaved with two restriction endonucleases EcoR I and Xho I , and the di gested products were separated and purified in low melting temperature agarose gel. The purified SAP2 and plasmid pcDNA3. 1 （＋）myc-HisC were recombined by T4DNA ligase, ligation products were transformed into competent cell, Escherichia coli TOP10. Transformed colonies were screened by Amp＇ LB plate, then recombined plasmids were isolated and identified by restriction endonuclease cutting and DNA sequencing. Results Identified by agarose gel electrophoresis, the target gene SAP2 obtained by PCR amplification had the same molecular size as predicted. It was indicated that recombined plasmids contained inserted SAP2 gene fragment by restriction endonuclease cutting analysis, the sequencing data also indicated that inserted SAP2 gene had correct DNA sequence and orientation according to DNA sequence of Candida albicans SC5314. Conclusion Eukaryotic expression plasmid pcDNA3. 1 （ ＋ ）myc HisC is used conveniently and effectively for constructing pcDNA3.1/SAP2. It can stimulate antigen re-presenting cell. It suggests that recombined plasmid pcDNA3. 1/SAP2 has high immunogenicity and immune response, and can be used as gene vaccine candidate. |
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| Keywords: | Candida albicans aspartic proteinases SAP2 gene recombinant plasmid gene vaccine |
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