CAJ | 학술논문

目的通过基因克隆在巴斯德毕赤酵母中表达人自身抗原Sm B'.方法 PCR扩增Sm B'基因,与酵母表达载体pPIC9k重组,构建表达质粒pPIC9k-Sm B'.用电穿孔法转化酵母菌Sm D1168,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(WB)鉴定,并用免疫酶斑点法和免疫印迹法(immunoblot,IBT)检测与评价分析30例系统性红斑狼疮(SLE)患者、30例混合结缔组织病患者和30名健康体检者血清中抗-Sm B'水平差异.结果 PCR产物约700 bp,与预期657 bp接近,pPIC9k-Sm B'重组阳性克隆测序结果与核酸数据库报道完全一致,双酶切鉴定正确,表达产物Sm B'的相对分子质量约32 000,WB法证实表达产物具有天然Sm B'分子的免疫原性,阴性对照菌未见目的表达条带.免疫酶斑点法检测阳性率为46.7%(42/90),IBT的检测阳性率为51.1%(46/90);2种方法检测结果一致性较佳(Kappa值=0.911 2,P<0.01).结论 Sm B'在巴斯德毕赤酵母中分泌表达成功,这为Sm B'试剂盒研究奠定了基础.
목적 통과기인극륭재파사덕필적효모중표체인자신항원Sm B'.방법 PCR확증Sm B'기인,여효모표체재체pPIC9k중조,구건표체질립pPIC9k-Sm B'.용전천공법전화효모균Sm D1168,재MD평판상사선중조극륭,용G418쾌속사선고고패전화자,양성극륭경갑순유도표체후,배양상청액용십이완기류산납-취병희선알응효전영(SDS-PAGE)화단백질인적법(WB)감정,병용면역매반점법화면역인적법(immunoblot,IBT)검측여평개분석30례계통성홍반랑창(SLE)환자、30례혼합결체조직병환자화30명건강체검자혈청중항-Sm B'수평차이.결과 PCR산물약700 bp,여예기657 bp접근,pPIC9k-Sm B'중조양성극륭측서결과여핵산수거고보도완전일치,쌍매절감정정학,표체산물Sm B'적상대분자질량약32 000,WB법증실표체산물구유천연Sm B'분자적면역원성,음성대조균미견목적 표체조대.면역매반점법검측양성솔위46.7%(42/90),IBT적검측양성솔위51.1%(46/90);2충방법검측결과일치성교가(Kappa치=0.911 2,P<0.01).결론 Sm B'재파사덕필적효모중분비표체성공,저위Sm B'시제합연구전정료기출.
Objective To clone and express human autoantigen Sm B'in methylotrophie yeast Pichia Pagtoris.Methods The gene Sm B' was cloned bv PCR The PCR product wag inserted into the vector pPIC9k.The recombinant plasmid pPIC9k.Sm B' was transformed into yeast Sm D1168 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 methan01.Supematants after induction were analyzed by SDS-PAGE and western blot.Sera collected from thirty patients with SLE.thirty patients with mixed connective tissue disease(MCTD)and thirty healthy volunteers were detected by immunodot and immunoblot.Results The PCR product wag about 700 bD in size which Wag in accordance with predicted 657 bp.The pPIC9k-Sm B'showed the same seqencing result with GenBank's report and restriction enzyme analysis confirmed our prediction.The pPIC9k-Sm B' positive clone produced a 32 000 protein which had natural immunogenicitv of human autoantigen Sm B'by SDS-PAGE and western blot.The positive rate of immunodot and IBT were 46.7%(42/90)and 51.1%(46/90),respectively.The agreement between immunodot and IBT was very close(Kappa value=0.911 2,P＜0.01).Conclusion Successfully cloning and expression of human autoantigen Sm B' in methylotmphic yeast Pichia Pagtoris hid a foundation for further research work.