摘要
目的在巴斯德毕赤酵母中表达舍格伦综合征A抗原（SSA60）并建立胶体金快速检测方法。方法表达质粒pPIC9k—SSA60用电穿孔法转化酵母菌SMD1168，在MD平板上筛选重组克隆，用G418快速筛选高拷贝转化子，阳性克隆经甲醇诱导表达后，培养上清用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）和斑点免疫金渗滤法（DIGFA）鉴定。结果表达产物SSA60的分子量约60000，高拷贝巴斯德毕赤酵母转化菌的表达水平明显高于低拷贝的，表达量占分泌总蛋白60％以上，产物浓度为600～950mg／L。DIGFA检测85份舍格伦综合征（SS）患者血清和30份正常人血清，抗SSA检测阳性率为95．3％（81／85），特异度为100％（30／30）。与德国欧蒙ENA酶斑点法对比检测的符合率为96．5％（111／115），差异无统计学意义（P〉O．05）。结论SSA60在巴斯德毕赤酵母中获得高效分泌表达，DIGFA简便、快速、准确，可用于临床自身免疫病的诊断。
Objective To express human Sjogren＇s syndrome antigen A60（SSA60） in methylotrophic yeast Pichia Pastoris and establish a new method with dot immunogold filtration assay（DIGFA）. Methods The recombinant plasmid pPIC9k- SSA60 was transformed into yeast SMDl168 by electroporation. The positive clones were screened in MD plates. The high copy number transformants were rapidly selected by using G418 and were induced by methanol. Supernatants after induction were analyzed by SDS-PAGE and DIGFA. Results The pPIC9k-SSA60 positive clone produced a 60 000 protein which had natural immunogenicity of human autoantigen SSA60 by SDS-PAGE and DIGFA. The expression product of high copy number transformants amounted to 600- 950 mg/L,over 60% of the total secreted protein. Sensitivity and specificity of DIGFA were 95.3 % （81/85） and 100% （30/30） ,respectively. The coincidence rate of the results of simultaneous determination of serum for autoantibody SSA60 with DIGFA and dot-ELISA was 96.5% （111/115） and no significant statistical difference was found（P〉0. 05）. Conclusion Human autoantigen SSA60 gave successfully high-level secreted expression in methylotrophic yeast Pichia pastoris. DIGFA,more simple more rapid and accurate ,could be applied for diagnosis on autoimmunune diseases.
出处
《现代检验医学杂志》
CAS
2009年第1期37-39,共3页
Journal of Modern Laboratory Medicine
基金
福建省青年人才科技创新基金资助项目（No.2002J060）.
