白假丝酵母菌天冬氨酸蛋白酶Sap2多克隆抗体的制备、纯化及鉴定

Preparation,purification and identification of polyclonal antibody against secretory aspartyl proteinase 2 of Candida albicans

目的制备白假丝酵母菌天冬氨酸蛋白酶Sap2多克隆抗体,并对其进行鉴定。方法提取白假丝酵母菌基因组DNA为模板,经聚合酶链反应(PCR)获取SAP2目的基因;双酶切SAP2基因与原核表达载体pMAL-c2x(+),构建pMAL-c2x/SAP2重组质粒;在大肠埃希菌BL21(DE3)感受态细胞中经IPTG诱导表达出可溶性的融合蛋白;用可溶性Sap2免疫小鼠制备抗血清,间接酶联免疫吸附试验(ELISA)检测抗血清效价,亲和层析法纯化抗血清后,利用Western免疫印迹(Western Blot)检测抗体特异性。结果该研究成功制备了抗Sap2多克隆抗体,抗体效价>1∶51 200;Western Blot检测结果表明,该抗体可特异性识别Sap2蛋白。结论纯化后的Sap2作为抗原免疫小鼠,有较好的抗原性和免疫原性,可成功制备多克隆抗体,并具有高特异性,为快速检测侵袭性白假丝酵母菌感染奠定了基础。

Objective To prepare and identify polyclonal antibodies against secretory aspartyl proteinase 2（Sap2） of Candida albicans. Methods Candida albicans genomic DNA was extracted as template, target gene fragment SAP2 was obtained by standard PCR amplification~ SAP2 and plasmids pMAL-c2x （ ＋ ） were cleaved with two re- striction endonucleases, and digested products were joined, then the recombinant plasmid pMAL-e2x/SAP2 was con- structed; Sap2 was expressed as soluble form after induced by IPTG in Escherichia coli strain BL21 （DE3）. Nine BALB/c mice were immunized with soluble Sap2, so as to produce antiserum against Sap2. The titer of antiserum was determined by indirect enzyme-linked immunosorbent assay（ELISA）. After purified by ProteinG affinity chro- matography, anti-Sap2 polyelonal antibody was resolved by SDS-PAGE and its specificity was determined by West- ern Blot. Results Anti-Sap2 polyclonal antibody was successfully prepared and the titer was ）1 ： 51 200. The anti- body could specifically recognize Sap2 by Western Blot analysis. Conclusion Soluble Sap2 protein can be used as an- tigen to immunize animal. It has better antigenicity and immunogenicity. Polyclonal antibody against Sap2 can be prepared by animal immunization and expressed high specificity, which has laid the foundation for development of rapid diagnosis of invasive Candida albicans infection.