白念珠菌ORF19.6012-MYC融合菌株的构建及鉴定

摘要

目的 构建白念珠菌ORF19.6012-MYC融合菌株.方法 利用In-Fusion试剂盒构建插入片段,采用醋酸锂转染法将片段同源重组至SN152基因组DNA中,采用PCR以及Western blotting方法进行验证,对验证为阳性的菌株进行生长曲线的测定及菌丝诱导实验.结果 经验证,成功构建ORF19.6012-MYC融合菌株,且ORF19.6012-MYC融合菌的生长增殖能力、液体菌丝形成能力与亲本菌一致.结论 采用同源重组的方法成功构建稳定表达的ORF19.6012-MYC融合菌株.%Objective To construct ORF19.6012-MYC strain in Candida albicans.Methods The recombinant fragment composed of the upstream,downstream of the ORF19.6012 and the MYC-SAT1 flipper was constructed by In-Fusion kit.Then the fragment was homologously recombined into the genomic DNA of SN152 by lithium acetate transfection.The MYC tag was added to the C terminus of the open reading frame of ORF19.6012.PCR and Western blotting were used to verify the positive strain.Finally,the growth curve and hyphal formation ability of the positive strain were determined.Results ORF19.6012-MYC fusion strain was successfully constructed.The growth and proliferation ability of ORF19.6012-MYC strain and the ability of hyphal formation were consistent with those of the parental strain.Conclusion The ORF19.6012-MYC strain was successfully constructed and would be used in the following experiments.