CAJ | 학술논문

目的利用RT-QuIC研究带有D178N突变的朊蛋白的自我转化能力.方法以带有人PRNP基因的质粒为模板,通过定点突变,构建了带有D178N突变的人PRNP基因,利用原核表达系统表达并纯化了带有N端及N端缺失的D178N朊蛋白.分别将此蛋白作为底物,利用RT-QuIC体外纤维形成实时监测系统,对各种突变蛋白体外纤维形成进行了观察和比较,并对形成纤维蛋白的蛋白酶抗性进行了初步分析和比较.结果正确构建了D178N突变的PRNP基因;在RT-QuIC的反应条件下,缺失N端的带有D178N突变的人朊蛋白,即使在不存在prpSc种子的情况下,在体外也能够发生自我转化,且形成的纤维具有蛋白酶抗性;而带有N端的D178N突变的朊蛋白,在RT-QuIC的反应条件下不发生自我转化.结论朊蛋白N端对D178N突变的朊蛋白自发纤维形成具有一定影响,这为探索和研究遗传型克雅病的致病机制提供了重要线索.
목적 이용RT-QuIC연구대유D178N돌변적원단백적자아전화능력.방법 이대유인PRNP기인적질립위모판,통과정점돌변,구건료대유D178N돌변적인PRNP기인,이용원핵표체계통표체병순화료대유N단급N단결실적D178N원단백.분별장차단백작위저물,이용RT-QuIC체외섬유형성실시감측계통,대각충돌변단백체외섬유형성진행료관찰화비교,병대형성섬유단백적단백매항성진행료초보분석화비교.결과 정학구건료D178N돌변적PRNP기인;재RT-QuIC적반응조건하,결실N단적대유D178N돌변적인원단백,즉사재불존재prpSc충자적정황하,재체외야능구발생자아전화,차형성적섬유구유단백매항성;이대유N단적D178N돌변적원단백,재RT-QuIC적반응조건하불발생자아전화.결론 원단백N단대D178N돌변적원단백자발섬유형성구유일정영향,저위탐색화연구유전형극아병적치병궤제제공료중요선색.
Objective To study the conversion of mutant D178N prion protein in RT-QuIC assay.Methods The D178N mutant prion PRNP was generated by the method of single site mutation.The mutant PRNP gene was inserted into plasmids of pET24.The full and N-truncated recombinant human prion proteins were expressed and purified.The fibril formations of these proteins were real-time monitored by the method of RT-QuIC.The ability to resist proteinase K (PK) of these fibrils was analyzed.Results We succeed to construct human PrP-D178N plamids.The N-truncated human prion protein with D178N (PrP90-231-D178N) can convert spontaneously in RT-QuIC,while full length of human prion D178N protein(PrP23-231-D178N) fails to convert spontaneously.The spontaneously generated fibril has been domenstrated it is partily PK-resistant.Conclusion The N-terminal of prion protein (23-90) plays an important role for the D178N mutant protein spontaneously conversion,which provide the clues for study the pathogenesis of genetic CJD.