白念珠菌mp65基因真核质粒的构建及免疫原性研究

目的构建以白念珠菌甘露糖蛋白65(MP65)编码基因mp65为目的基因真核重组表达质粒,并对其免疫原性进行分析。方法PCR法自白念珠菌标准菌株ATCC64550中获取mp65基因,分别插入真核表达载体pcDNA3.1中,将真核表达经质粒大抽提并定量后免疫BALB/c小鼠,ELISA法检测抗体产生水平,流式检测细胞免疫。结果PCR法克隆出全长为1140bp的mp65基因,构建出pcDNA3.1-mp65真核表达质粒,pcDNA3.1-mp65免疫小鼠能诱导产生效价为1∶800的IgG抗体,同时诱导CD4+T和CD8+T细胞百分含量增高。结论白念珠菌真核表达质粒pcDNA3.1-mp65成功构建并能诱导小鼠的体液免疫和细胞免疫。

Construction of the eukaryotic expression plasmid for mp65 gene of Candida albicans

To construct the eukaryotic expression plasmid of the mp65 gene in Candida albicans and to analyze its immunogenicity, this gene was firstly amplified by using the PCR technique from the standard strain ATCC 64550 mice, and then inserted to eukaryotic expression vector peDNA3.1. The eukaryotic expression plasmid was extracted and was used to immunize mice, while the antibodies produced against MP65 protein as well as the cell mediated immune responses were detected by ELISA and flow cytometry respectively. In this way, the rap65 gene with a molecular size of 1140 bps was harvested by PCR and the eukaryotic expression plasmid pcDNA3, 1-rap65 was constructed successfully. This recombinant plasmid used to immunize BALB/c mice could induce the production of IgG antibodies with a serum titer of 1 ： 800. Also, the percentages of CD4^＋ and CD8^＋ T lymphocytes were elevated. It is evident that the humoral and cell-mediated immune responses can be induced by the immunization of mice with the eukaryotic expression plasmid pcDNA3.1-mp65.