【作者】 朱飞舟；

【导师】 张灼华；

【作者基本信息】 中南大学 ， 细胞生物学， 2007， 博士

【摘要】 帕金森病(Parkinson’s Disease,PD)是一种最常见的神经退行性运动障碍疾病,该病的特征是依赖年龄的静止性震颤,肌肉强直,躯体麻痹,经多巴胺的前体左旋多巴(L-dopa)治疗后症状减轻。在年龄大于65岁的德国Bavarian人群中,PD的发病率为0.71%;在年龄大于50岁的新加坡人中,PD的发病率大约为0.30%。PD的病理学特征为黑质致密部多巴胺能神经元缺失,以及在剩余的多巴胺能神经元中出现泛素阳性的Lewy小体。迄今在染色体上已发现至少有九个位点与家族性PD相关,分别为PARK1-PARK9,PARK13,其中PARK1与PARK4为同一位点4q21。已克隆的PD相关基因有7个,其中α-synuclein、UCH-L1、LRRK2和Omi/HtrA2基因的突变可导致常染色体显性(autosomal dominant,AD)遗传PD,parkin、PINK1和DJ-1基因的突变则导致常染色体隐性(autosomal recessive,AR)PD。LRRK2基因有51个外显子,编码的蛋白多达2527个氨基酸,包含ANK、LRR、ROC、COR、Kinase和WD40等多个结构域。已经发现的PD相关突变在LRRK2的各个结构域中均有分布,尤以位于Kinase结构域的G2019S最为常见。LRRK2的分子量大约280KD,在脑和其它器官以及细胞系HEK293T中均可表达,功能尚不清楚。已有的研究表明,LRRK2R1441C、G2019S、I2020T等PD相关突变导致LRRK2激酶活性上升,细胞生活力下降,说明这些突变对细胞是有毒性的。而且,LRRK2参与调节神经元突起的形态建成,并在突触囊泡蛋白的极性分布中起重要的作用。Anti-LRRK2抗体是研究LRRK2功能重要的、必须的工具。通过DNAstar的Protean软件分析LRRK2的氨基酸序列,选择了三段长度在100个氨基酸左右、抗原性强、亲水性好的多肽片段,将其编码的核苷酸序列构建至原核表达载体pGEX-4T-2,原核表达和纯化得到了GST融合蛋白,然后免疫新西兰雄兔,获得了效价高、特异性好的anti-LRRK2兔多克隆抗体。抗体用途分析实验表明,该抗体可用于Western Blotting,免疫组织化学,免疫细胞染色等实验。利用自制的anti-LRRK2抗体,绘制出了LRRK2在小鼠脑中的分布图,发现LRRK2在鼠脑中是广泛表达的,但各神经解剖学部位间有重要的不同;Western Blotting的结果表明LRRK2在大鼠脑各部位均可表达;在HEK293细胞中LRRK2与线粒体标志物细胞色素C可以共定位。另外,序列比对的结果表明LRRK2与LRRK1的氨基酸序列是高度同源的。更多还原

【Abstract】 Parkinson’s disease (PD) is the most frequent progressive neurodegenerative movement disorder characterized by age-dependent resting tremor, muscular rigidity, akinesia and relieved temporarily by administration of L-dopa. The prevalence of PD is 0.71% in a German Bavarian population of individuals older than 65 years, and is 0.30% in Singaporeans aged 50 years and older. The pathological hallmarks of PD patients include progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta as well as the presence of ubiquitin-positive Lewy neuritis and Lewy bodies in the remaining neurons. Nine loci in the chromosome have been found to be associated with family PD including PARK1-PARK9 and PARK13 in which PARK1 and PARK4 share the same locus. Mutations in at least seven genes are individually linked to familial forms of PD, including autosomal dominant mutations in a-synuclein, UCH-L1, LRRK2, Omi/HtrA2 and autosomal recessive mutations in parkin, PINK1, and DJ-1.LRRK2 gene has 51 exons. The LRRK2 protein has 2527aa, including ANK, LRR, ROC, COR, Kinase, WD40 six domains. The discovered mutations associated with PD are distributed in all the domains of LRRK2 and the most common one is G2019S. The MW of LRRK2 is 280 KD. It is expressed in brain, other organs and cell line HEK293T with unclear functions. Some research have showed that mutations associated with PD, such as R1441C, G2019S, I2020T, lead to increasing of LRRK2 kinase activity and decreasing of cell activity, indicating that these mutations are toxic to cells. Moreover, LRRK2 plays a role in neurite process morphology and polarity of synaptic vesicles (SV) protein.Anti-LRRK2 antibody is a necessary and important tool of LRRK2 functional research. To prepare anti-LRRK2 antibody, I analyzed LRRK2 protein sequence by DNAstar/Protean software and selected 3 polypeptides which are about 100aa, high antigenicity and good hydrophilicity. Then, the CDS of the selected 3 polypeptides were constructed to pGEX-4T-2 and the GST fusion protein were purified. With the purified GST fusion protein, the rabbits were immunized and a high titer, high specificity anti-LRRK2 rabbit polyclonal antibody was gotten. Functional analysis of anti-LRRK2 showed that it was available in immunohistochemistry, immunostaining and Western Blotting.With the anti-LRRK2 antibody, the distribution of LRRK2 in mouse brain was mapped. It was showed that LRRK2 protein is broadly distributed throughout the mouse brain, but shows important regional differences. Also, the result of Western blotting showed that LRRK2 is expressed in all sections of rat brain. In HEK293, LRRK2 is co-localized with cytochrome C. In addition, the result of alignment showed that amino acids sequences of LRRK2 and LRRK1 are highly homogenous.更多还原