白念珠菌果糖二磷酸醛缩酶重组蛋白的制备及鉴定

摘要：目的：制备有免疫原性的白念珠菌果糖二磷酸醛缩酶（fructosebisphosphate aldolase, Fba1)重组蛋白。 方法：以白念珠菌C1标准株基因组DNA为模板，用PCR法扩增Fba1 DNA序列，与克隆载体pMD18-T连接、测序，再与原核表达载体pET28a(+)连接，构建成重组表达质粒pET28a(+)/Fba1，转化大肠埃希菌BL21(DE3)，IPTG诱导重组融合蛋白表达，亲和层析柱纯化重组蛋白。用抗His标签的单克隆抗体鉴定融合蛋白，并用确诊为侵袭性白念珠菌病（ICAI）、且抗白念珠菌烯醇化酶抗体阳性的患者血清进行抗原性鉴定。 结果：Fba1 DNA序列与GenBank中的序列一致。在大肠埃希菌中获得白念珠菌Fab1重组蛋白的高效表达，表达产物以可溶性蛋白为主，最终蛋白质得率为7.6 mg/g湿菌。经免疫印迹鉴定，重组蛋白与抗His标签的单克隆抗体和确诊ICAI患者血清呈特异性反应，显示出良好的抗原反应性。 结论：成功制备了具有良好抗原反应性的重组蛋白，为建立特异性诊断ICAI的新方法打下基础。

Preparation and identification of recombinant fructose-bisphosphate aldolase of Candida albicans

Objective To prepare recombinant fructose-bisphosphate aldolase(Fba1) of Candida albicans and identify its immunogenicity.Methods The full-length coding sequence of Fba1 was obtained by PCR from genomic DNA of C.albicans C1 strain,and cloned into pMD18-T vector.The sequencing demonstrated the amplified DNA sequence of Fba1 to be correct.The Fba1 sequence was connected with the prokaryotic expression vector pET28a(+) to construct the recombinant expression vactor pET28a(+)/Fba1 which was then transferred int...