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目的:制备特异性光滑假丝酵母耐药基因CDR1(CgCDR1)的多克隆抗体,为深入研究CgCDR1在介导光滑假丝酵母耐药中的作用奠定基础。方法:根据生物信息学分析预测CgCDR1蛋白的二级结构、亲水性及抗原性等理化特性,以这些分析预测为依据,经过同源性检索后设计出一段由14个氨基酸组成的多肽,免疫新西兰兔,制备多克隆抗体。用ELISA、Western blot方法检测该抗体的效价和特异性,并用Western blot方法检测CgCDR1在配对光滑假丝酵母菌中的表达情况。结果:ELISA法检测所制备抗体的效价达1∶5 000;Western blot结果显示该抗体能与CgCDR1蛋白特异结合。CgCDR1在氟康唑耐药的光滑假丝酵母菌中的表达量明显高于配对的氟康唑敏感的光滑假丝酵母菌。结论:制备的CgCDR1多克隆抗体具有较高的效价和良好的特异性,该多克隆抗体可用于CgCDR1的免疫活性鉴定,为深入研究该蛋白在光滑假丝酵母耐药中的作用奠定了基础。
OBJECTIVE: To prepare polyclonal antibodies against Candida glabrata-resistant gene CDR1 (CgCDR1) and lay a foundation for further study on the role of CgCDR1 in the mediation of Candida-resistant Candida. Methods: Based on bioinformatics analysis, physicochemical properties such as secondary structure, hydrophilicity and antigenicity of CgCDR1 protein were predicted. Based on the analysis and prediction, a 14 amino acid peptide was designed after homologous search, New Zealand rabbits to prepare polyclonal antibodies. The titer and specificity of the antibody were detected by ELISA and Western blot. The expression of CgCDR1 in Candida glabrata was detected by Western blot. Results: The titer of the prepared antibody was 1: 5000 by ELISA. The result of Western blot showed that the antibody could specifically bind with CgCDR1 protein. The expression of CgCDR1 in fluconazole-resistant Candida glabrata was significantly higher than that of paired fluconazole-sensitive Candida glabrata. CONCLUSION: The prepared polyclonal antibody against CgCDR1 has high titer and good specificity. The polyclonal antibody can be used to identify the immunological activity of CgCDR1, which lays the foundation for further study on the role of CgCDR1 in C.albicans resistance .