斜紋夜蛾Caspase-3、Caspase-6的基因克
發(fā)布時(shí)間：2018-08-25 18:50
【摘要】：細(xì)胞凋亡是機(jī)體細(xì)胞在正常生理或病理狀態(tài)下,遵循自身程序發(fā)生的一種自主的、程序化的死亡過程,它受到一系列基因、蛋白的嚴(yán)密調(diào)控,依賴于天冬氨酸的半胱氨酸蛋白酶(Caspase)在細(xì)胞凋亡途徑中起著不可替代的關(guān)鍵酶作用。迄今,在哺乳動(dòng)物中已經(jīng)鑒定至少有14種Caspase,雙翅目昆蟲果蠅已鑒定有7種Caspase,鱗翅目昆蟲Caspase家族近年來逐漸被鑒定和分析。鱗翅目昆蟲斜紋夜蛾(Spodoptera litura)細(xì)胞凋亡方面的研究已經(jīng)有較多的報(bào)道,但有關(guān)Caspase方面的研究工作較少,為開展Caspase的鑒定以及在細(xì)胞凋亡通路中作用等研究,本實(shí)驗(yàn)克隆了斜紋夜蛾Caspase-3、Caspase-6的基因,原核表達(dá)、純化了 Caspase-3、Caspase-6蛋白并分析其活性,以期為深入解析斜紋夜蛾細(xì)胞凋亡的分子機(jī)制提供有用的資料和數(shù)據(jù)。1.Sl-caspase-3基因克隆,表達(dá)純化及活性分析采用PCR方法擴(kuò)增出斜紋夜蛾Caspase-3基因(Sl-caspase-3),其ORF長(zhǎng)846bp,編碼281個(gè)氨基酸,預(yù)測(cè)蛋白相對(duì)分子質(zhì)量為31.8kDa,等電點(diǎn)為6.55,含有Caspase特征性的QACRG五肽序列,結(jié)構(gòu)分析顯示Sl-caspase-3沒有DED或CARD結(jié)構(gòu)域,屬于效應(yīng)Caspase,同源蛋白比對(duì)分析發(fā)現(xiàn)Sl-caspase-3與家蠶ICE-2相似度達(dá)到56%。將Sl-caspase-3基因插入pET22b載體,獲取陽(yáng)性質(zhì)粒pET-22b-caspase-3,并轉(zhuǎn)化感受態(tài)細(xì)胞Rosetta-gami(DE3),用異丙基硫代-β-D-半乳糖苷(IPTG)誘導(dǎo),Caspase-3得到表達(dá),SDS-PAGE和Western blotting檢測(cè)顯示,表達(dá)產(chǎn)物為Sl-caspase-3完整蛋白。利用鎳柱、分子純化系統(tǒng)對(duì)蛋白進(jìn)行分離純化,純化產(chǎn)物用作酶活性測(cè)定。酶活性離體測(cè)定顯示,活化后的Sl-caspase-3蛋白可以分別切割斜紋夜蛾效應(yīng)Caspase Sl-caspase-1(哺乳動(dòng)物Caspase-3的同源物)以及起始Caspase Sl-caspase-5(哺乳動(dòng)物Caspase-9的同源物)的突變型蛋白Sl-caspase-l-C178A、Sl-caspase-5-C310A,這提示Sl-caspase-3與斜紋夜蛾其他Caspase之間有關(guān)聯(lián),預(yù)示與其他Caspase間存在級(jí)聯(lián)反應(yīng),但具體機(jī)制尚不清楚,需要進(jìn)一步研究。2.S1-caspase-6基因克隆、表達(dá)純化及活性分析成功克隆了斜紋夜蛾Caspase-6基因(Sl-caspase-6,其ORF長(zhǎng)1569bp,編碼522個(gè)氨基酸,預(yù)測(cè)蛋白相對(duì)分子質(zhì)量為60.3kDa,等電點(diǎn)為6.71,結(jié)構(gòu)分析顯示Sl-caspase-6前端含有DED區(qū),具有起始Caspase特征。利用原核系統(tǒng)表達(dá)Sl-caspase-6 蛋白,SDS-PAGE 及 Western blotting 分析顯示,Sl-caspase-6 大量表達(dá)后發(fā)生Caspase常見的降解現(xiàn)象。為此,我們采取截短法方式,表達(dá)Sl-caspase-6的CASc功能域(大小亞基區(qū)域),以此構(gòu)建了 pET-22b-Sl-caspase-6-N224載體,表達(dá)和純化了 Sl-caspase-6-N224 蛋白產(chǎn)物。SDS-PAGE 電泳顯示,Sl-caspase-6-N224蛋白可自我激活并且切割成分子大小為23.1kDa和12.2kDa兩條帶;Western blotting分析表明,Sl-caspase-6-N224重組蛋白能與6×His-tag抗體產(chǎn)生特異性反應(yīng)而被檢測(cè)到。此外,以人工Ac-IEVD-AFC作為熒光底物,Sl-caspase-6-N224蛋白對(duì)此底物有酶解作用,顯示其具有Caspase活性。由于Ac-IEVD-AFC是Caspase-8特異性的的底物,這提示斜紋夜蛾Sl-caspase-6與哺乳動(dòng)物細(xì)胞Caspase-8可能具有相似功能,更進(jìn)一步說明Sl-caspase-6與哺乳動(dòng)物Caspase-8在結(jié)構(gòu)和功能上的相似性,都屬于上游起始Caspase。根據(jù)細(xì)胞凋亡分子和凋亡機(jī)制的保守性,推測(cè)斜紋夜蛾細(xì)胞凋亡途徑中可能存在類似于哺乳動(dòng)物的死亡受體信號(hào)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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.4
[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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.4
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