人自身抗原U1RNP 70K的酵母重组分泌表达及斑点免疫金渗滤检测法的建立  

Secreted expression of recombinant human autoantigen U1RNP 70K in methylotrophic yeast Pichia Pastoris and establishment of dot immunogold filtration assay

作　　者：杨湘越[1] 张惠菊[1] 徐秀云[1] 兰小鹏[1] 朱忠勇[1] 

机构地区：[1]南京军区福州总医院解放军临床检验医学研究所

出　　处：《医学研究生学报》2011年第9期908-912,共5页Journal of Medical Postgraduates

基　　金：福建省青年人才科技创新基金(2002J060)

摘　　要：目的人自身抗原U1RNP 70K通常采用组织提取法或原核表达获得。文中用基因克隆技术,在毕赤酵母中表达人U1RNP 70K自身抗原,并建立斑点免疫金渗滤检测法。方法 PCR扩增U1RNP 70K基因,与酵母表达载体pPIC9k重组,构建表达质粒pPIC9k-U1RNP 70K。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 b lot)鉴定。用所表达的蛋白包被硝酸纤维素膜,建立检测U1RNP抗体的斑点免疫金渗滤法(dot immuogold filtrationassay,D IGFA)。结果 PCR产物约为1300 bp,接近于预期的1314 bp,pPIC9k-U1RNP 70K重组阳性克隆测序结果与Gen-Bank中的U1RNP 70K序列完全一致,双酶切鉴定正确,表达产物U1RNP 70K的相对分子质量约70 000。W estern b lot证实表达产物具有天然U1RNP 70K分子的免疫原性,阴性对照菌无目的条带表达。D IGFA的检测结果与欧蒙酶免疫斑点法的阳性符合率为94.74%(54/57),阴性符合率为98.00%(49/50),总符合率为96.26%(103/107)。2种方法检测结果无统计学显著差异(P=0.617)。结论在毕赤酵母中成功地高分泌表达了U1RNP 70K蛋白,并建立了简便、快速、准确检测U1RNP抗体的DIGFA方法。Objective Human autoantigen U1RNP 70K is normally obtained by tissue extraction or prokaryotic expression.In this study,we cloned and expressed human autoantigen U1RNP 70K in methylotrophic yeast Pichia Pastoris and established a new dot immunogold filtration assay（DIGFA）.Methods The gene of U1RNP 70K was cloned by PCR,and the recombinant plasmid pPIC9k-U1RNP 70K was transformed into yeast SMD1168 by electroporation.The positive clones were screened in MD plates.The high copy number transformants were rapidly selected by G418 and induced by methanol.The post-induction supernatants were analyzed by SDS-PAGE and Western blot.DIGFA was established for the detection of U1RNP using the protein-coated nitric acid cellulose membrane.Results The PCR product was about 1300 bp in size,close to the predicted 1314 bp.The sequencing results of pPIC9k-U1RNP 70K showed no difference from those in the GenBank report and restriction enzyme analysis confirmed our prediction.The products of U1RNP 70K showed a relative molecular mass of about 70 000,with natural immunogenicity of human autoantigen U1RNP 70K.The positive and negative rates of concordance of the results of DIGFA with those of enzyme immunodot were 94.74%（54/57） and 98.00%（49/50）,respectively,with no statistically significant differences between the two methods（P=0.617）.Conclusion A high-level expression of human autoantigen U1RNP 70K was obtained in the methylotrophic yeast Pichia Pastoris,and a new DIGFA was successfully established for the simple,rapid and accurate detection of U1RNP.

关 键 词：自身抗原 U1RNP70K 克隆 表达 巴斯德毕赤酵母 斑点免疫金渗滤法 

分 类 号：R392.11[医药卫生—免疫学]