家蚕基因BmNPC1的克隆及功能分析

发布时间：2018-03-11 18:21

本文选题：家蚕　切入点：BmNPC1　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：人类C型尼曼匹克病(Niemann-Pick disease type C,NP-C)是一种罕见的,进展性的且不可逆的疾病,为常染色体隐性遗传,常导致神经系统功能缺失甚至早逝。95%患者是由于NPC1基因突变导致的胆固醇代谢障碍,另5%是NPC2基因突变所致。作为导致C型尼曼匹克病的主要因素,NPC1参与细胞内的胆固醇运输,编码位于溶酶体和核内体膜上的13次跨膜蛋白。NPC1突变的细胞内胆固醇异常堆积于核内体/溶酶体中。NPC1基因已经被发现有较多的功能,主要是:NPC1参与运输细胞内胆固醇;NPC1蛋白又被证实有跨膜分子泵活性;在针对于埃博拉病毒的研究中,NPC1蛋白被发现与埃博拉病毒感染细胞相关。以果蝇为例的昆虫中NPC1参与胆固醇运输外还被发现与蜕皮以及精子形成相关,且有两个直系同源物,NPC1a和NPC1b。而关于家蚕中NPC1基因功能尚无报道。为探索其在家蚕中功能,克隆得到了家蚕NPC1基因,并分析了其表达情况。之后制备了该基因的多克隆抗体。最后通过dsRNA干涉技术对BmNPC1基因的功能进行进一步的研究。主要研究结果如下:1.家蚕Bm NPC1基因全长克隆及信息分析利用家蚕基因组数据库和NCBI等数据库,对NPC1进行基本信息分析及比对并设计引物,BmNPC1的全长通过RACE技术获得。BmNPC1基因cDNA全长4591bp,其中ORF阅读框为3702bp,位于第三号染色体上。编码1233个氨基酸,蛋白大小为137.44kD,含有一个胆固醇结合结构域,且该蛋白13次跨膜。选取各物种的NPC1蛋白序列,构建系统发生树,结果显示BmNPC1与果蝇的NPC1亲缘关系最近。2.家蚕Bm NPC1的表达分析取家蚕五龄三天的所有组织和从四龄至熟蚕整个时期的家蚕血液,并用来提取cDNA作为模板,通过qRT-PCR检测BmNPC1的表达情况。结果表明,BmNPC1在五龄三天所有组织中表达,且在精巢、卵巢以及血液和马氏管中表达量比较高。在血细胞中的表达量与时期相关,当家蚕处于变态期时该基因大量表达。3.家蚕Bm NPC1抗体的制备及检测BmNPC1的多克隆抗体通过以下四步制备:构建重组的原核表达载体pET32,通过IPTG诱导蛋白的大量表达,在包涵体中的蛋白的纯化,以及5次小鼠免疫。最终获得了BmNPC1的小鼠多克隆抗体。利用ELISA测定抗体效价,之后Western blot检测获得的条带大小与BmNPC1预测的蛋白分子量一致。家蚕血液和家蚕细胞系(SID-1)的免疫荧光实验表明,与哺乳动物相同(NPC1蛋白定位于溶酶体膜和核内体膜),BmNPC1蛋白同样出现在细胞质中。表明抗体制备有效,可用的,为下一步实验做了良好的基础。4.利用dsRNA干涉技术研究Bm NPC1基因功能设计并合成双链干涉片段,细胞水平上通过双链干涉片段干涉SID-1细胞,FilipinⅢ染色发现细胞中胆固醇发生明显聚集,推测家蚕BmNPC1与细胞的胆固醇运输相关;个体水平上双链干涉片段注射蚕体,一定量的家蚕蜕皮推迟,另有一部分没有完成蜕皮并死亡,推测该基因与家蚕蜕皮过程相关。
[Abstract]:Niemann-Pick disease type CNP-C) is a rare, progressive and irreversible disease with autosomal recessive inheritance. Some 95% of patients with neurological dysfunction or even premature death are due to cholesterol metabolism due to mutations in the NPC1 gene and 5% to mutations in the NPC2 gene. NPC1 is involved in the transport of cholesterol in cells as a major factor leading to type C Neimanpik disease. The cellular cholesterol abnormal accumulation of 13 transmembrane protein. NPC1 mutations on lysosome and intranuclear membranes has been found to have many functions in the nucleosome / lysosome. It was found that the protein of 1: NPC1 was involved in the transport of cholesterol and NPC1 protein and had transmembrane molecular pump activity. In the Ebola virus study, NPC1 protein was found to be associated with Ebola virus-infected cells. In Drosophila, for example, NPC1 involved in cholesterol transport was also found to be associated with molting and spermatogenesis. There are two direct homologues, NPC1a and NPC1b.However, the function of NPC1 gene in Bombyx mori has not been reported. In order to explore its function in silkworm, the NPC1 gene of Bombyx mori was cloned. Then the polyclonal antibody of the gene was prepared. Finally, the function of BmNPC1 gene was further studied by dsRNA interference technique. The main results were as follows: 1.Full-length cloning of BmNPC1 gene of Bombyx mori and. Information analysis and use of silkworm genome database and NCBI database, The full length of NPC1 gene BmNPC1 was analyzed and compared with that of primer BmNPC1. The full length of BmNPC1 gene cDNA was obtained by RACE technique. The ORF reading frame was 3702 BP, which was located on chromosome 3 and encoded 1233 amino acids. The protein is 137.44kD. it contains a cholesterol binding domain, and the protein transmembrane 13 times. The phylogenetic tree was constructed by selecting the NPC1 protein sequences of each species. The results showed that the relationship between BmNPC1 and Drosophila NPC1 was the most recent .2.The expression of BmNPC1 in Bombyx mori was analyzed. All tissues of Bombyx mori and the blood of silkworm from 4th instar to mature silkworm during the whole period from 4th instar to mature silkworm were collected and used to extract cDNA as template. The expression of BmNPC1 was detected by qRT-PCR. The results showed that the expression of BmNPC1 was higher in spermary, ovary, blood and Markov tube than in all tissues on the 3rd day of fifth instar. The expression of BmNPC1 in blood cells was correlated with the period. When Bombyx mori is in metamorphosis stage, the BmNPC1 antibody of Bombyx mori and the polyclonal antibody to detect BmNPC1 are prepared in the following four steps: construct the recombinant prokaryotic expression vector pET32, and induce protein expression by IPTG. The protein in inclusion body was purified and immunized for 5 times. Finally, the polyclonal antibody of BmNPC1 was obtained. The antibody titer was determined by ELISA. The size of the band detected by Western blot was the same as that of the protein predicted by BmNPC1. The immunofluorescence assay of silkworm blood and silkworm cell line (SID-1) showed that, The same NPC1 protein was located in the cytoplasm of lysosomal membrane and nuclear membrane, indicating that the preparation of antibodies was effective and useful. 4. Using dsRNA interference technique to study the function of Bm NPC1 gene design and synthesis of double strand interference fragment. At the cellular level, we found that the cholesterol concentration in SID-1 cells was significantly aggregated by double-strand interference fragment interference (SID-1) staining, which suggested that BmNPC1 was related to cholesterol transport in silkworm cells, while at individual level, double-strand interference fragments were injected into silkworm body. A certain amount of silkworm molting was delayed, while others did not complete molting and died. It is speculated that the gene is related to the molting process of Bombyx mori.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596.1;Q78

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