烟曲霉果胶裂解酶A重组蛋白的原核表达及抗原性分析  

Prokaryotic expression and antigenic analysis of recombinant pectate lyase A of Aspergillus fumigatus

作　　者：张国勇[1,2] 刘倩[1,2] 李芳秋[2] 史利宁[2] 胡毓安[2] 李伟[2] 商秀娟[2] 

机构地区：[1]南京师范大学生命科学学院,南京210097 [2]南京军区南京总医院临床中心实验科,南京210002

出　　处：《临床检验杂志》2015年第2期108-110,共3页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81302536);南京军区医学科技创新重点课题(10Z027);江苏省科技支撑计划-社会发展项目(BE2009673)

摘　　要：目的制备烟曲霉果胶裂解酶A(pectate lyase A,Ply A)重组蛋白质并鉴定其抗原性。方法对编码烟曲霉ply A基因的DNA序列进行优化和人工合成,序列分析验证,然后连接至表达载体p ET-28a(+),并转化大肠埃希菌(E.coli)BL21(DE3)。用异丙基硫代半乳糖苷(IPTG)诱导重组蛋白质表达,SDS-PAGE电泳分析重组蛋白质,并用Talon亲和层析柱纯化,western blot分析重组蛋白质与抗组氨酸标签蛋白(His-tag)单克隆抗体和侵袭性曲霉病患者血清中相应抗体的反应性。结果克隆出经过修饰改良的、适用于原核细胞表达的烟曲霉ply A基因的DNA序列;获得相对分子量约35 300的Ply A重组蛋白质;western blot结果显示该蛋白质可被抗His-tag抗体识别,并与曲霉病患者血清中抗体发生特异性免疫反应。结论成功表达烟曲霉Ply A重组蛋白质,初步证明该蛋白质的抗原性良好。Objective To prepare recombinant pectate lyase A（ Ply A） of Aspergillus fumigatus and identify its antigenicity. Methods The DNA encoding ply A of Aspergillus fumigatus was optimized and synthesized. The sequence of DNA was inserted into p ET-28a（ ＋） vector,which was transformed into E. coli BL21（ DE3）. The recombinant protein was expressed by IPTG induction.Following the analysis of SDS-PAGE the expressed protein was purified by Talon metal affinity resins. The reactions of the recombinant protein with anti-His-tag antibody and the antibody in the serum from invasive aspergillosis patients were analyzed by western blot. Results The DNA sequence of encoding ply A of Aspergillus fumigatus was modified to be suitable for expression in prokaryocyte and was cloned. The recombinant Ply A with molecular mass 35 300 was expressed in E. coli. Western blot showed that the recombinant protein could be recognized by anti-His-tag antibody and specifically reacted to the antibody in the serum from patients with confirmed invasive aspergillosis. Conclusion The recombinant Ply A of Aspergillus fumigatus was successfully prepared and showed expected antigenicity.

关 键 词：烟曲霉 果胶酶A 原核表达 

分 类 号：Q78[生物学—分子生物学]