斜纹夜蛾Caspase-3、Caspase-6的基因克

发布时间：2018-08-25 18:50

【摘要】：细胞凋亡是机体细胞在正常生理或病理状态下,遵循自身程序发生的一种自主的、程序化的死亡过程,它受到一系列基因、蛋白的严密调控,依赖于天冬氨酸的半胱氨酸蛋白酶(Caspase)在细胞凋亡途径中起着不可替代的关键酶作用。迄今,在哺乳动物中已经鉴定至少有14种Caspase,双翅目昆虫果蝇已鉴定有7种Caspase,鳞翅目昆虫Caspase家族近年来逐渐被鉴定和分析。鳞翅目昆虫斜纹夜蛾(Spodoptera litura)细胞凋亡方面的研究已经有较多的报道,但有关Caspase方面的研究工作较少,为开展Caspase的鉴定以及在细胞凋亡通路中作用等研究,本实验克隆了斜纹夜蛾Caspase-3、Caspase-6的基因,原核表达、纯化了 Caspase-3、Caspase-6蛋白并分析其活性,以期为深入解析斜纹夜蛾细胞凋亡的分子机制提供有用的资料和数据。1.Sl-caspase-3基因克隆,表达纯化及活性分析采用PCR方法扩增出斜纹夜蛾Caspase-3基因(Sl-caspase-3),其ORF长846bp,编码281个氨基酸,预测蛋白相对分子质量为31.8kDa,等电点为6.55,含有Caspase特征性的QACRG五肽序列,结构分析显示Sl-caspase-3没有DED或CARD结构域,属于效应Caspase,同源蛋白比对分析发现Sl-caspase-3与家蚕ICE-2相似度达到56%。将Sl-caspase-3基因插入pET22b载体,获取阳性质粒pET-22b-caspase-3,并转化感受态细胞Rosetta-gami(DE3),用异丙基硫代-β-D-半乳糖苷(IPTG)诱导,Caspase-3得到表达,SDS-PAGE和Western blotting检测显示,表达产物为Sl-caspase-3完整蛋白。利用镍柱、分子纯化系统对蛋白进行分离纯化,纯化产物用作酶活性测定。酶活性离体测定显示,活化后的Sl-caspase-3蛋白可以分别切割斜纹夜蛾效应Caspase Sl-caspase-1(哺乳动物Caspase-3的同源物)以及起始Caspase Sl-caspase-5(哺乳动物Caspase-9的同源物)的突变型蛋白Sl-caspase-l-C178A、Sl-caspase-5-C310A,这提示Sl-caspase-3与斜纹夜蛾其他Caspase之间有关联,预示与其他Caspase间存在级联反应,但具体机制尚不清楚,需要进一步研究。2.S1-caspase-6基因克隆、表达纯化及活性分析成功克隆了斜纹夜蛾Caspase-6基因(Sl-caspase-6,其ORF长1569bp,编码522个氨基酸,预测蛋白相对分子质量为60.3kDa,等电点为6.71,结构分析显示Sl-caspase-6前端含有DED区,具有起始Caspase特征。利用原核系统表达Sl-caspase-6 蛋白,SDS-PAGE 及 Western blotting 分析显示,Sl-caspase-6 大量表达后发生Caspase常见的降解现象。为此,我们采取截短法方式,表达Sl-caspase-6的CASc功能域(大小亚基区域),以此构建了 pET-22b-Sl-caspase-6-N224载体,表达和纯化了 Sl-caspase-6-N224 蛋白产物。SDS-PAGE 电泳显示,Sl-caspase-6-N224蛋白可自我激活并且切割成分子大小为23.1kDa和12.2kDa两条带;Western blotting分析表明,Sl-caspase-6-N224重组蛋白能与6×His-tag抗体产生特异性反应而被检测到。此外,以人工Ac-IEVD-AFC作为荧光底物,Sl-caspase-6-N224蛋白对此底物有酶解作用,显示其具有Caspase活性。由于Ac-IEVD-AFC是Caspase-8特异性的的底物,这提示斜纹夜蛾Sl-caspase-6与哺乳动物细胞Caspase-8可能具有相似功能,更进一步说明Sl-caspase-6与哺乳动物Caspase-8在结构和功能上的相似性,都属于上游起始Caspase。根据细胞凋亡分子和凋亡机制的保守性,推测斜纹夜蛾细胞凋亡途径中可能存在类似于哺乳动物的死亡受体信号通路。
[Abstract]:Apoptosis is an autonomous and programmed process of cell death in normal physiological or pathological conditions. It is closely regulated by a series of genes and proteins. Caspase, a caspase-dependent enzyme, plays an irreplaceable key role in the pathway of cell apoptosis. At least 14 caspases have been identified in mammals, 7 caspases have been identified in Diptera Drosophila, and the Lepidoptera Caspase family has been identified and analyzed in recent years. In order to identify Caspase and study its role in apoptosis pathway, Caspase-3 and Caspase-6 genes of Spodoptera litura were cloned and expressed in prokaryotic cells. The proteins of Caspase-3 and Caspase-6 were purified and their activities were analyzed in order to provide useful information and data for further understanding the molecular mechanism of apoptosis of Spodoptera litura. The caspase-3 gene (Sl-caspase-3) of Spodoptera litura was cloned, expressed, purified and analyzed by PCR. The ORF of the gene was 846 BP long, encoding 281 amino acids. The predicted relative molecular weight of the protein was 31.8 kDa, the isoelectric point was 6.55, and it contained the QACRG pentapeptide sequence characterized by Caspase. Structural analysis showed that there was no DED or CARD in Sl-caspase-3. Sl-caspase-3 gene was inserted into pET22b vector to obtain the positive plasmid pET-22b-caspase-3, and transformed into the receptive cell Rosetta-gami (DE3), induced by isopropylthio-beta-D-galactoside (IPTG), and Caspase-3 was expressed. SDS-P-P-P was used as the expression vector. AGE and Western blotting assay showed that the expressed product was a complete protein of Sl-caspase-3. The protein was isolated and purified by a nickel column and purified by a molecular purification system. The purified product was used for enzyme activity assay. In vitro enzyme activity assay showed that the activated Sl-caspase-3 protein could cleave Caspase Sl-caspase-1 (Casp-caspase-1) of Spodoptera litura, respectively. Sl-caspase-l-C178A, Sl-caspase-5-C310A, the homologue of ase-3 and the mutant protein Sl-caspase-5 (the homologue of Caspase-9 in mammals), suggest that Sl-caspase-3 is associated with other caspases of Spodoptera litura, suggesting a cascade reaction with other caspases, but the mechanism is not clear and needs further study. 2. S1-caspase-6 gene (Sl-caspase-6, 1569 BP long, encoding 522 amino acids) was cloned. The relative molecular weight of the predicted protein was 60.3 kDa, and the isoelectric point was 6.71. Structural analysis showed that the front end of Sl-caspase-6 contained the DED region and had the characteristics of initial caspase. The expression of Sl-caspase-6 protein was analyzed by SDS-PAGE and Western blotting. The results showed that the degradation of Caspase was common after the overexpression of Sl-caspase-6. Therefore, we constructed the vector pET-22b-Sl-caspase-6-N224 by truncating the CASc domain of Sl-caspase-6. SDS-PAGE electrophoresis showed that the Sl-caspase-6-N224 protein could self-activate and cleave two bands with molecular sizes of 23.1 kDa and 12.2 kDa. Western blotting analysis showed that the recombinant protein of Sl-caspase-6-N224 could react specifically with 6 *His-Ac tag antibody and was detected by using artificial IEVD-AFC as fluorescence. The substrate, Sl-caspase-6-N224, has enzymatic hydrolysis to this substrate, indicating its caspase activity. Ac-IEVD-AFC is a specific substrate of Caspase-8, suggesting that Sl-caspase-6 of Spodoptera litura may have similar functions with Caspase-8 of mammalian cells, and further demonstrates that Sl-caspase-6 and Caspase-8 of mammals have similar structures and functions. According to the conservative nature of apoptotic molecules and apoptotic mechanism, it is speculated that there may be death receptor signaling pathway similar to that of mammals in apoptotic pathway of Spodoptera litura.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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