白念珠菌β-葡萄糖苷酶2的原核细胞表达、鉴定及免疫反应性  

Prokaryotic expression,identification and immunoreactivity of Candida albicans β-glucosidase 2

作　　者：贺政新[1] 冉向阳[1] 陈晶[1] 王宪灵[1] 侯天文[1] 

机构地区：[1]解放军白求恩国际和平医院检验实验科,石家庄050082

出　　处：《临床检验杂志》2014年第1期59-61,共3页Chinese Journal of Clinical Laboratory Science

基　　金：河北省自然科学基金(C2010001882)

摘　　要：目的构建白念珠菌β-葡萄糖苷酶2(BGL2)的原核细胞表达质粒,诱导其表达后加以鉴定,并初步判定重组蛋白质的免疫反应性。方法 PCR法获取白念珠菌SC5314的bgl2基因,插入原核细胞表达载体pET30a中,以重组质粒转染感受态细胞大肠埃希菌(E.coli)BL21,经IPTG诱导表达后,SDS-聚丙烯酰胺凝胶电泳(PAGE)检测蛋白质的表达情况,梯度透析法对包涵体蛋白质复性,用抗His标签单克隆抗体鉴定。用临床侵袭性念珠菌病(IC)患者血清判定重组蛋白质的免疫反应性。结果成功构建原核细胞表达质粒pET30a-bgl2,诱导后的重组蛋白质以包涵体形式表达,经梯度透析复性成功。重组蛋白质能被抗His标签抗体识别,并与IC患者血清有良好的反应性。结论用原核细胞表达体系成功表达了白念珠菌BGL2,该蛋白质具有良好的免疫反应性,为进一步研究其在IC诊断中的作用打下基础。Objective：To construct and identify prokaryotic expression plasmids of Candida albicans β-glucosidase 2 gene （ bgl2 ） and examine the immunoreactivity of the recombinant protein following inducible expression. Methods：The full length coding sequence of bgl2 was amplified by PCR and cloned into the prokaryotic expression vector pET30a. The 6×His tagged protein was induced by IPTG and the recombinant vector was transfected into competent E. coli BL-21（DE3）. The expressed protein purified by His-trap-HP metal affinity resins was analyzed by SDS-PAGE. The immunoreactivity of the recombinant protein was evaluated by western blot using the sera from the patients with invasive candidiasis （IC）. Results：The full-length bgl2 gene was cloned from Candida albicans SC5314 genome and pET30a-bgl2 plasmid was successfully constructed. The recombinant protein BGL2 was highly expressed in E. coli in the form of inclusion body and then renatured by gradient dialysis. BGL2 recombinant protein was recognized by specific monoclonal antibody to His-tag and showed high immunoreactivity with the sera from IC patients. Conclusion：Recombinant BGL2 protein was successfully expressed by prokaryotic expression system and the strong immunoreactivity of the recombinant protein should be helpful for the research on the diagnosis of IC.

关 键 词：白念珠菌 原核细胞表达 β-葡萄糖苷酶2 侵袭性念珠菌病 

分 类 号：R446.5[医药卫生—诊断学]