人自身抗原SSA52的酵母重组分泌表达及其斑点免疫金渗滤法的建立

Secreted Expression of Recombinant Human Autoantigen SSA52 in Methylotrophic Yeast Pichia pastoris and Establishment of Dot Immunogold Filtration Assay

人自身抗原SSA52通常采用组织提取法或原核表达获得,存在诸多问题。通过基因克隆技术,在毕赤酵母中表达人自身抗原SSA52,并建立斑点免疫金渗滤法。采用RT-PCR扩增SSA52基因,与酵母表达载体pPIC9k重组,构建表达质粒pPIC9k-SSA52。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,培养上清液用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blot)鉴定,并建立斑点免疫金渗滤法(dot immuogold filtration assay,DIGFA),进行初步临床应用对比研究。结果显示,RT-PCR产物约为1 400 bp,与预期1 428 bp接近,pPIC9k-SSA52重组阳性克隆测序结果与基因库核酸数据库报道完全一致,双酶切鉴定正确,表达产物SSA52的相对分子质量约52 kD。Western blot法证实表达产物具有天然SSA52分子的免疫原性,阴性对照菌未见目的表达条带。DIGFA与欧蒙酶免疫斑点法的阳性符合率为95.2%(120/126),阴性符合率为94.0%(47/50),总符合率为94.9%(167/176);两种方法检测结果无统计学显著差异(P>0.05)。说明SSA52在毕赤酵母中分泌表达成功,建立的DIGFA简便、快速、准确。

Human autoantigen SSA52 was obtained by tissue extracting or expression in E.coli.We cloned and expressed human autoantigen SSA52 in methylotrophic yeast Pichia pastoris and established a new dot immunogold filtration assay（DIGFA）.The gene SSA52 was cloned by RT-PCR.The PCR product was inserted into the vector pPIC9k.The recombinant plasmid pPIC9k-SSA52 was transformed into yeast SMD1168 by electroporation.The positive clones were screened in MD plates.The high copy number transformants were rapidly selected using G418 and induced by methanol.Supernatants after induction were analyzed by SDS-PAGE and Western blot.The clinical specimen for SLE,SS,RA,SSc and healthy individuals were detected by DIGFA and enzyme immunodot.The PCR product was about 1 400 bp in size,which was in accordance with predicted 1 428 bp.The pPIC9k-SSA52 showed the same sequencing result with GenBank’s report and restriction enzyme analysis confirmed prediction.The pPIC9k-SSA52 positive clone produced a 52 kD protein which had natural immunogenicity of human autoantigen SSA52 by SDS-PAGE and Western blot.The positive and negative concordance of DIGFA were 95.2%（120/126）and 94.0%（47/50）,respectively.There was no significant statistical difference between DIGFA and enzyme immunodot（P0.05）.Successfully cloning and expression of human autoantigen SSA52 in methylotrophic yeast Pichia pastoris were obtained,which proved that DIGFA is simple,rapid and accurate.