【作者】 董辰方；

【导师】 董小平；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2008， 博士

【摘要】 可传播性海绵状脑病（Transmissible spongiform encephalopathies,TSEs）即朊病毒病（prion diseases）,是一类罕见的致死性神经系统退行性疾病,包括人类的克雅氏病（Creutzfeldt-Jakob disease,CJD）、GSS综合症（Gerstmann-Str（a|¨）ussler-Scheinker syndrome,GSS）、Kuru病、家族性致死性失眠症（fatal familialinsomnia,FFI）、绵羊和山羊的瘙痒病（scrapie）,牛及其它动物的海绵状脑病等,根据疾病的来源不同可分为自发形成、遗传性和医源性三种形式。本文分为三部分,第一部分我们利用原核表达系统表达了全长人PrP、各种截短的PrP以及各种PrP八肽重复区突变体,采用pull-down实验和免疫共沉淀方法探讨了PrP和微管蛋白之间的相互作用以及PrP蛋白多肽链中与微管蛋白（tubulin）相互作用的区域。此外,我们通过共沉淀法、比浊法及透射电镜法检测不同的PrP对微管蛋白聚合的影响。第二部分我们优化了链霉素对PrP^Sc沉淀的方法,并分析了该方法对PrP^Sc检测的敏感性。第三部分我们在分子水平证实了PrP与GFAP间的相互作用,并进一步确定PrP蛋白中与GFAP蛋白的相互作用区域。第一部分:PrP对微管蛋白聚合的影响研究我们利用pull-down,免疫共沉淀试验确定全长PrP蛋白PrP23-231可以与微管蛋白发生相互作用。我们进一步检测了PrP蛋白与微管蛋白的相互作用位点,结果表明缺失突变体N端23-91 aa、23-50 aa和51-91 aa均可以与微管蛋白发生相互作用,而PrP C端91-231 aa不能与微管蛋白发生相互作用,确定PrP蛋白与微管蛋白的相互作用位点位于PrP N端23-50 aa和八肽重复区51-91 aa。为了检测PrP与tubulin的相互作用是否影响微管的聚合,我们用体外微管聚集实验、共沉淀实验及透射电镜法对其进行检测。结果显示,N端的PrP23-91及C端的PrP91-231均不影响微管蛋白的聚合,而全长的PrP23-231能够有效的抑制微管蛋白的聚合。提示PrP对微管蛋白聚合的影响是全长PrP依赖性的。为了检测含有不同八肽重复区数目的PrP突变体与微管蛋白的结合能力,我们利用ELISA检测含不同八肽重复数目的PrP（PG0,5,9,12）与包被的微管蛋白的结合能力,结果显示与PG5的作用结果相比,含9个及12个八肽重复的PG9和PG12结合微管蛋白的能力明显增强,而八肽重复区完全缺失的PG0对微管蛋白的结合能力明显较低,这些结果提示PrP蛋白八肽重复序列的存在数量直接影响PrP与微管蛋白的结合效应,八肽重复数目越多,其结合作用越明显。为了检测PrP八肽重复区突变体对微管蛋白聚合的影响,我们用体外微管聚集实验、共沉淀实验及透射电镜法对其进行检测。结果显示,野生型PrP及PrP八肽重复区突变体均能影响微管蛋白的聚合。PrP八肽重复序列插入突变体（PG9和PG12）较野生型PrP（PG5）均有更强的抑制作用,而缺失八肽重复序列的PrP（PG0）虽然与微管蛋白的结合能力明显减弱,然而对微管聚合的抑制作用仍旧存在,甚至强于PG5、PG9和PG12。这些结果提示,八肽重复序列的数目不仅与PrP和微管蛋白的结合力有关,而且在PrP对微管动力学的调节过程中也起着重要作用。只有正确数目的八肽重复序列的PrP才能有效的平衡对微管聚合的调节。第二部分:链霉素增加PrP^Sc检测的敏感性我们优化了链霉素对PrP^Sc沉淀的方法。首先评估了不同量的链霉素对PrP^Sc的沉淀效果,结果显示链霉素浓度与沉淀中PrP^Sc的量呈剂量依赖性。此外,我们检测了溶液的pH值是否能影响链霉素沉淀效果,结果发现PrP^Sc的沉淀效果受溶液pH值影响,在中性或弱酸性的条件下,链霉素能够稳定有效的沉淀PrP^Sc;在碱性条件下,随着pH值的增加,链霉素对PrP^Sc的沉淀效果随之减弱。为了检测样品经链霉素处理后是否增加PrP^Sc在Western blot检测的敏感性,我们将PrP^Sc贮存液分别加入到含有终浓度为60 mmol/L链霉素的裂解液中。结果显示无论是在低浓度或大容积的条件下,链霉素沉淀法均可显著的提高PrP^Sc在Western blot检测的敏感性。为了评估链霉素沉淀法对脑组织及体液中PrP^Sc的检测效果,我们将PrP^Sc贮存液分别加入到含有终浓度为60 mmol/L链霉素的正常仓鼠脑组织匀浆上清、人脑脊液和尿液中。结果显示,不同样品的PrP^Sc的反应条带均可被有效检测到。提示链霉素沉淀法能够有效的富集脑组织、脑脊液以及尿液中PrP^Sc。此外,我们也用链霉素沉淀法对fCJD病人脑组织的PrP^Sc进行检测,结果显示该方法能够有效的检测出病人脑组织中的PrP^Sc。第三部分:PrP及其缺失突变体与GFAP体外相互作用的初步研究我们通过免疫共沉淀实验显示无论在正常或羊瘙痒因子263K感染的仓鼠脑组织中都存在GFAP-PrP复合物,提示脑组织中的GFAP与PrP^C和PrP^Sc可能发生相互作用。为了进一步获得PrP与GFAP相互作用的直接证据,利用原核表达系统及体外翻译系统表达了全长的GFAP蛋白、PrP蛋白以及各种缺失突变体。利用pull-down及免疫共沉淀实验从分子水平证实PrP在体外可与GFAP发生特异性的分子间相互作用。我们进一步检测了PrP蛋白与GFAP蛋白的相互作用位点,结果表明PrP蛋白C端91-231 aa可以与GFAP蛋白发生相互作用,而N端23-91aa不能与GFAP蛋白发生相互作用,确定PrP蛋白与GFAP蛋白的相互作用位点位于PrP C端91-231 aa。此外,我们的研究显示经PK消化后的PrP^Sc仍能够与GFAP蛋白发生分子间相互作用。更多还原

【Abstract】 Transmissible spongiform encephalopathies（TSEs）,prion diseases,are rare degenerative neurological disorders that afflict human beings,including Creutzfeldt-Jakob disease（CJD）,Gerstmann-Str（a｜¨）ussler-Scheinker syndrome（GSS）, Kuru,and fatal familial insomnia（FFI）,sheep and goat（scrapie）,cattle（bovine spongiform encephalopathy,BSE）,and other animals.They may have a sporadic, inherited or acquired origin.This study contains three individual parts,including investigation of the effect of PrP on microtublule polymerization,the analyses of sensitive detection of PrP^Sc by Western blot assay based on streptomycin sulfate precipitation and the study of molecular interaction between prion protein and GFAP both in native and recombinant forms in vitro.PartⅠ:Analysis of the effects of PrP on microtubule polymerization in VitroUsing pull-down and co-immunoprecipitation assay,a remarkable interaction between the full-length His-PrP23-231 and tubulin was identified.We further mapped the regions within PrP binding with tubulin with pull-down and co-immunoprecipitation assay,and demonstrated that the N terminus peptides containing PrP 23-91,PrP23-50 and PrP51-91 could interact with tublin,while the C terminus peptide PrP91-231 failed.It demonstrated that the interaction regions within PrP with tublin located at the N terminus peptide 23-50 aa and octapeptide repeat region 51-91 aa.To test whether the interaction between PrP and tubulin influenced the assembling of microtubules from tubulin in vitro,microtubule assembly assay, sedimentation test and morphological observation of transmission electron microscopy were performed.Our results showed that only PrP in the context of full-length peptide is concerned with the modulation of microtubule dynamics as a tubulin-sequestering protein,while its N- or C-terminal segments seem not to affect microtubule polymerization in vitro.To evaluate the binding activities with tubulin among various PrPs with different numbers of octapeptide repeats,various PrPs were employed into a tubulin-coated ELISA.Compared with that of PG5,the reactions of PG9 and PG12 showed stronger binding activities with tubulin,whereas that of PG0 was obviously weaker.The results indicated that the numbers of octapeptide repeats within PrP directly affected the binding activity with tubulin and the binding activity became stronger along with the increase of numbers of octapeptide repeats.To investigate the effectiveness of the numbers of octapeptide repeats within PrP on microtubule dynamics,microtubule assembly assay,sedimentation test and morphological observation of transmission electron microscopy were performed. Compared with PG5,PG9 and PG12 induced much higher turbidities,showing a number-dependent manner.Interestingly,although PG0 showed weaker binding activity with tubulin,an unexpectedly strong increase of solution turbidity was repeatedly observed in the preparation of PG0,which was even higher than that of PG9 and PG12.Thus,the numbers of octapeptide repeats may not only be associated with binding activity of PrP to tubulin,but also play an important role in regulating inhibitive effect of PrP on microtubule polymerization.Only the correct numbers of octapeptide repeats can efficiently balance the regulating activity of PrP to the tubulin polymerization.PartⅡ:Sensitive detection of PrP^Sc by Western blot assay based on streptomycin sulfate precipitationWe optimized the Western blot assay for PrP^Sc with a precipitation procedure of streptomycin sulfate.In order to probe the optimal working concentration of streptomycin sulfate,different quantities of streptomycin sulfate were subjected into the preparations containing PrP^Sc stock sample.Western blot analyses identified a clearly streptomycin sulfate dose-dependant manner of PrP^Sc distribution in fractions of the pellets.In addition,we evaluated the influence of pH value on the effectiveness of streptomycin precipitation for PrP.The results showed that the precipitating activity of streptomycin for PrP was quite efficient and stable under neutral or weakly acidic condition,but dropped down along with the increase of pH value.To seek whether treatment of PrP^Sc with streptomycin sulfate increased the identifying sensitivity in Western blots,different amounts of hamster PrP^Sc stock sample were incubated with streptomycin sulfate in lysis buffer.The results demonstrated that streptomycin precipitation increased markedly the detective sensitivity of PrP^Sc,regardless in low concentration or in large volume.To address the precipitating activities of streptomycin sulfate on PrP^Sc in brain tissue or body fluids,hamster PrP^Sc stock sample was spiked into hamster PrP^C stock sample,CSF and urine,respectively.Contrast to the preparations without streptomycin sulfate that no positive signal was observed,the specific PrP^Sc reactive signals were obviously detected in all preparations.In addition,the PrP^Sc from a human brain tissue of familiar Creutzfeldt Jacob Disease（fCJD） was efficiently precipitated with streptomycin sulfate. PartⅢ:Molecular interaction between prion protein and GFAP both in native and recombinant forms in vitroIn the present study,remarkable GFAP-PrP^Sc or GFAP-PrP^C complexes were separately detected in the brain homogenates of 263K（Scrapie）-infected or normal hamsters by co-immunoprecipitation assay.To get more exact molecular evidences for interaction between prion protein（PrP） and GFAP,various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing and in-vitro translation system.Using pull down and co-immunoprecipitation assays,reliable molecular interaction between PrP and GFAP was observed,and proteinase K（PK）-digested PrP^Sc molecules were confirmed to be able to bind the recombinant GFAP specifically as well.The region within PrP that was responsible for interaction with GFAP was narrowed to PK-resistant core of PrP（i.e.aa91-231）.更多还原