斜纹夜蛾亲免素A和亲免素10的分子克隆及表达

发布时间：2018-04-01 11:23

本文选题：斜纹夜蛾　切入点：亲免素A　出处：《云南大学》2015年硕士论文

【摘要】：亲免素(Cyclophilins, Cyps)是广泛存在于生物体内的保守蛋白家族且都具有肽酰脯氨酸顺反异构酶(Peptidyl-prolyl cis-trans isomerases, PPIase)的活性。亲免素在一系列的生物学过程中发挥重要作用,如辅助蛋白折叠、参与细胞凋亡、信号转导、HIV-1病毒蛋白组装等过程。近年来相关亲免素的研究主要集中在哺乳动物中,而在斜纹夜蛾中的研究还是首次。本研究所获得的一些结果为进一步探索在昆虫免疫反应中亲免素A和亲免素10起到的作用提供重要的信息。提取斜纹夜蛾的血淋巴,反转录成cDNA,以cDNA为模版克隆得到两个基因：SpliCypA和SpliCyplO。SpliCypA开放阅读框ORF的大小为498bp,编码165个aa,其理论分子量为17.62kDa。SpliCyp10开放阅读框ORF的大小为490bp,编码161个aa,其理论分子量为17.89kDa。斜纹夜蛾CypA的ORF编码氨基酸的序列分别与大家鼠CypA、非洲爪蟾CypA、人CypA、家蚕CypA、粘虫CypA的ORF编码的氨基酸序列相比较,其同源性分别为74.4%、73.9%、76.8%、83.6%、和95.8%。得到了这些基本信息后,我们构建了SpliCypA的原核表达载体pET32a-SpliCypA,并通过南京巴傲德生物科技有限公司制备得到了SpiCypA的抗体。在此基础上,成功地构建了真核表达载体pIZT/V5-His-SpliCypA, Western blot检测到SpliCypA在四种昆虫细胞(Bml2、Spli22、Sf9、High Five)中的均能表达,其融合蛋白的大小为21.29kDa。免疫荧光显示CypA蛋白在High Five细胞的细胞质中。用真核表达质粒pIZT/V5-His-SpliCypA转染High Five细胞后,利用RNA i技术,沉默High Five细胞中的CypA,用制备得到的抗体进行检测,我们发现实验组pIZT/V5-His-CypA-siRNA-22和pIZT/V5-His-CypA-siRNA-37比对照组表达蛋白量均减弱。同时我们还构建SpliCyp10真核表达载体pIZT/V5-His-SpliCyplO,其融合蛋白的分子量为21.75kDa。pIZT/V5-His-SpliCyp10转染昆虫细胞Sf9和Spil221,96h提取细胞的总蛋白,Western blot结果显示该质粒能在这两种昆虫细胞中有效表达,免疫荧光显示Cyp10蛋白分布在细胞质当中,靠近在细胞核的位置。
[Abstract]:Cyclophilins (Cyps) is a family of conserved proteins widely found in organisms and has the activity of Peptidyl-prolyl cis-trans isomerases (PPIase).Immunophiles play an important role in a series of biological processes, such as protein folding, apoptosis, signal transduction, HIV-1 protein assembly and so on.In recent years, the studies on immunophiles are mainly concentrated in mammals, but this is the first study in Spodoptera litura.Some results obtained in this study provide important information for further exploring the role of immunophiles A and 10 in insect immune response.Extracting haemolymph of Spodoptera litura,Using cDNA as template, two genes were cloned into cDNA. the size of the two genes was 498bpand SpliCyplO.SpliCypA open reading frame ORF was 498bp. the theoretical molecular weight was 490bpof 17.62kDa.SpliCyp10 open reading frame ORF and 161aa, and its theoretical molecular weight was 17.89kDa.The amino acid sequences encoded by ORF of Spodoptera litura CypA were compared with the amino acid sequences of mouse CypA, Xenopus laevis CypA, human CypA, Bombyx mori CypA and CypA of Mythimyx armyworm, respectively. The homology of the amino acids encoded by ORF was 74.43.90.76. 83.6and 95.886, respectively.After obtaining these basic information, we constructed the prokaryotic expression vector pET32a-SpliCypA of SpliCypA, and prepared the antibody to SpiCypA through Nanjing Bahaode Biotechnology Co., Ltd.Immunofluorescence showed that CypA protein was present in the cytoplasm of High Five cells.After transfection of High Five cells with eukaryotic expression plasmid pIZT/V5-His-SpliCypA, RNA I technique was used to silence CypA in High Five cells. The results showed that the expression of pIZT/V5-His-CypA-siRNA-22 and pIZT/V5-His-CypA-siRNA-37 in the experimental group was lower than that in the control group.At the same time, we constructed SpliCyp10 eukaryotic expression vector pIZT / V5-His-SpliCyplo. The molecular weight of the fusion protein was 21.75kDa.pIZT/V5-His-SpliCyp10 transfected into insect Sf9 and Spil221 / 96h. The results of Western blot showed that the plasmid could be effectively expressed in these two insect cells.Immunofluorescence showed that Cyp10 protein was located in the cytoplasm, close to the nucleus.
【学位授予单位】：云南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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