【作者】 俞水亮；

【导师】 田波；

【作者基本信息】 武汉大学 ， 微生物学， 2005， 博士

【摘要】 可传播性海绵状脑病（TSE）是人和动物的一类罕见的、亚急性致死性神经退化疾病,包括疯牛病、羊搔痒病和近年来出现的人新型变异克雅氏病等。TSEs由细胞型朊病毒蛋白PrP^C转变成致病型朊病毒蛋白PrP^Sc引起,因此也称朊病毒病。人的朊病毒病有多种,其中10—15%可以遗传,并与人的朊病毒蛋白基因PRNP的突变有关,另有85%呈散发性。散发型朊病毒病的具体发生机制尚不清楚。已有的分析证明,PRNP基因的M129V和E219K多态性与朊病毒病的易感性关系密切,而且不同种族人群的M129V和E219K基因型和等位基因频率差异很大。 为了解我国PRNP基因型和等位基因频率的分布,我们调查了总计626例正常人的PRNP基因的M129V和E219K多态性,包括汉族、回族和维吾尔族三个民族的人群。我们发现129M/M纯合子基因型在三个民族中的分布明显不同,汉族129M/M基因型频率最高（98%）,其次是回族（85%）,最低为维吾尔族（60%）,均大大高于欧洲人群的129M/M基因型频率。而219E/E的基因型频率最高的是维吾尔族（98%）,其次为回族（96%）和汉族（90%）。这一结果提示我国人群可能较欧洲人群对朊病毒病更为易感。此外,我们还分析了另外两个较为少见的PRNP多态性位点:A117A和八肽重复区缺失一个八肽单位的突变（1-OPRD）。我们在3例维吾尔族个体中发现了A117A静息突变（gca→gcg）;在4例回族个体（2.0%）和1例汉族个体（0.5%）中发现了杂合的1-OPRD突变。 PRNP基因序列分析是发现遗传型朊病毒病的重要途径。我们用克隆测序的方法首次发现了插入3个额外八肽拷贝（72bp）的朊病毒新突变,并将其命名为PrP8G。PrP8G八肽重复区的基因序列是R1、R2、R2、R2a、R2、R2、R3、R4,与野生型序列（R1、R2、R2、R3、R4）对照,突变序列在R2和R3之间多出了R2a、R2、R2三个八肽拷贝,测序的结果还显示这一插入突变与129Met等位基因连锁,并以杂合形式存在。同样的插入在女孩母亲的基因组中也有发现,而其父亲和同父异母兄长的PRNP基因正常。随后,德国Grasbon-Frodl等在CJD病人的PRNP基因中也发现了类似的3个八肽的插入突变,提示PrP8G是一种致病突变。我们更多还原

【Abstract】 Transmissible spongiform encephalopathy(TSE) is a group of rare, sub-acute, fatal neurodegenerative diseases in human beings and animals, including scrapie, mad cow disease, Creutzfeld-Jacob disease(CJD) and so on. Based on the protein only hypothesis, TSE is caused by the conversion of a normal cellular prion protein (PrP~C) into a pathogenic, infectious isoform, which has been commonly referred to as scrapie prion protein (PrP~Sc). Human TSEs or prion diseases are described as infectious, inherited, and sporadic form. Approximately 10-15% of the human prion disease is inherited. The majority of the cases of human prion diseases (85%) occur sporadically by an unknown mechanism. Certain polymorphisms in the PRNP have been associated with the incidence or susceptibility of prion disease, such as the two most common polymorphisms, M129V and E219K. The distributions of M129V, E219K genotypes and alleles in various general populations show clear differences between Western Europeans and East Asian.To understand the genotypes and alleles frequencies of PRNP in Chinese populations and monitor the incidence of prion diseases in China, we screened a total number of 626 individuals, which represent three ethnic populations of China, Han, Hui, and Uyghur for M129V and E219K. The frequencies of Met/Met homozygote in these three groups differ significantly. The Han has a much higher frequency (98%) than Uyghur (85%) and Hui (60%). On the other hand, the frequencies of the Glu/Glu at residue 219 are higher in Uyghur (98%) and Hui (96%) than in Han (90%). This result show the higher frequency of 129Met homozygotes in Chinese than in Europeans, which may suggest that Chinese populations are at increased risk of developing prion diseases. We also investigated two other less common variants of -PRNP, a silent substitution at residue 117 (351A>G: A117A), and one octapeptide-repeat deletion mutation (1-OPRD) in the octapeptide-coding region. We found three Uyghur individuals with silent substitution at residue 117. Four Huiindividuals (2.0%) and one Han (0.5%) donor were found to be heterozygous for 1-OPRD.In the course of these studies, we found a healthy girl in Hui ethinic group with a novel three extra-repeat (72 bp) insertion within the octapeptide repeats coding region of PRNP, and named this mutation with PrP8G Sequencing results show that the most likely sequence of repeats in this case is Rl, R2, R2, R2a, R2, R2, R3, R4, with the extra repeats of R2a, R2, R2 inserted between normal R2 and R3 repeats. Identical mutation is also found in her mother but not in her father or a half-brother with a different mother. Three months later, Grasbon-Frodl et al. reported a similar mutation associated with a CJD patient. We also diagnosed a patient of fatal familial insomnia (FFI) by sequencing PRNP and found the D178N point mutation linked with the 129Met allele in this case. With exception of the Chinese FFI family that had been reported in Canada, this is the first FFI case found in China. Attempts have been made to collect as many blood samples and medical records as possible from these two family. Hopefully, we shall be able to establish a more extensive genealogy and follow the clinical development of each individual with the novel insertion mutation or Dl 78N point mutation.To investigate the pathogenic mechanism of the novel insertion, PCR was used to amplify the PrP23-23i gene from pMD-huPrP and pMD-huPrP8G plasmids, which contain the full length PRNP sequence of wild type and mutation respectively. The amplified fragments were inserted into pET30a and expressed in E.coli. The full-length mature huPrP and huPrP8G proteins were purified and refolded on column by using immobilized metal (Ni) affinity chromatography, based on the metal binding property of their unusual octarepeat sequences containing at least five tandem copies. The identification of purified proteins by SDS-PAGE and mass spectrum shows the purity is more than 95% and the Mr of huPrP and huPrP8G proteins are 22961 and 25292Da respectively. Different biochemical and biophysical properties were found between the wild type and mutant. Firstly, huPrP8G was much easier to aggregate than the wt. When the proteins were diluted from the same concentrations, the OD595 of huPrP8G increased much more than that of huPrP by 9.4 times in the buffer ofpH7.0 and by 17 times in pH7.5. Secondly, by SDS-PAGE without DTT, we found more dimers in huPrP8G than in huPrP. Furthermore, the mutant protein seemed to be more stable than the wt in acid circumstance when incubated in 37°C. These results indicate that huPrP8G mutant protein may be easy to converse into PrPSc-like structure compared with the wild type.更多还原