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

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.