论文部分内容阅读
目的:制备特异性白念珠菌外排泵(CaCDR1和CaCDR2)的多克隆抗体,为研究CaCDR1和CaCDR2在介导白念珠菌耐药中的作用奠定基础。方法:利用Pfam网络预测程序和Blastn、Blastx程序设计引物,采用PCR法获得白念珠菌SC5314基因组上486 bp长度的CDR1和CDR2目的基因,并将其克隆入pET-28a(+)质粒,构建重组表达质粒后,转化入大肠埃希菌BL21宿主菌内,在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下表达6×His融合蛋白;经镍柱纯化蛋白后,免疫新西兰兔,制备兔抗多克隆抗体。用ELISA及蛋白印迹法检测该抗体的效价和特异性。结果:ELISA法检测所制备抗体的效价最终定为1∶12 800,工作浓度定为1∶800;蛋白印迹法检测结果显示,抗体能与CaCDR1及CaCDR2蛋白特异性结合。结论:成功获得具有较高效价和良好特异性的CaCDR1、CaCDR2多克隆抗体,可用于白念珠菌CaCDR1、CaCDR2蛋白水平的鉴定。
OBJECTIVE: To prepare polyclonal antibodies against Candida albicans efflux pump (CaCDR1 and CaCDR2), and to lay the foundation for the study of CaCDR1 and CaCDR2 in mediating Candida albicans drug resistance. Methods: The 486 bp CDR1 and CDR2 genes of the Candida albicans SC5314 genome were amplified by PCR using the Pfam network prediction program and Blastn program and Blastx program, and cloned into pET-28a (+) plasmid to construct a recombinant The plasmid was transformed into Escherichia coli BL21 and expressed 6 × His fusion protein induced by isopropyl-β-D-thiogalactopyranoside (IPTG). The purified protein was purified by nickel column and immunized New Zealand Rabbit, rabbit anti-polyclonal antibody was prepared. The antibody titer and specificity were determined by ELISA and Western blotting. Results: The titer of the antibody prepared by ELISA was 1:12 800 and the working concentration was 1: 800. Western blotting showed that the antibody could specifically bind to CaCDR1 and CaCDR2. CONCLUSION: Polyclonal CaCDR1 and CaCDR2 antibodies with high titer and good specificity are successfully obtained for the identification of CaCDR1 and CaCDR2 protein levels in Candida albicans.