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。
Objective To prepare recombinant fructose-bisphosphate aldolase （ Fbal ） of Candida albicans and identify its immunogenicity. Methods The full-length coding sequence of Fbal 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 Fbal to be correct. The Fbal sequence was connected with the prokaryotic expression vector pET28a（ ＋ ） to construct the recombinant expression vactor pET28a（ ＋ ）/Fbal which was then transferred into E. coli BL21 （ DE3 ） and the recombinant fusion protein His6-Fbal was induced with IF＇TG. The His6-Fabl was further purified with TALON Metal Affinity Resin, and identified by Western blot using the monoclonal antibody against His6-tag. The immunogenicity of His6-Fabl was confirmed by western blot with the sera from the patients with systemic candidiasis. Results The sequence of prepared full-length DNA of C. albicans Fbal was coincident with that registered in GenBank. The recombinant protein His6-Fbal was highly expressed in E. coli with final yield of 7.6 mg/g in soluble form. Western blot showed that recombinant His6- Fbal protein could specifically react with monoclonal anti-his-tag antibody and the anti-enolase antibody in the sera from candidiasis pa- tients. Conclusion The recombinant fusion protein His6-Fbal with high immunogenicity was successfully prepared, and it should be helpful to develop a novel specific assay for diagnosis of invasive candidiasis.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2011年第5期375-377,共3页
Chinese Journal of Clinical Laboratory Science
基金
江苏省科技支撑计划-社会发展项目(BE2009673)
关键词
白念珠菌
果糖二磷酸醛缩酶
基因克隆
原核表达
Candida albicans
fructose-bisphosphate aldolase
gene cloning
prokaryotic expression
