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  本文選題：斜紋夜蛾　切入點：親免素A　出處：《云南大學》2015年碩士論文

【摘要】：親免素(Cyclophilins, Cyps)是廣泛存在于生物體內(nèi)的保守蛋白家族且都具有肽酰脯氨酸順反異構(gòu)酶(Peptidyl-prolyl cis-trans isomerases, PPIase)的活性。親免素在一系列的生物學過程中發(fā)揮重要作用,如輔助蛋白折疊、參與細胞凋亡、信號轉(zhuǎn)導、HIV-1病毒蛋白組裝等過程。近年來相關(guān)親免素的研究主要集中在哺乳動物中,而在斜紋夜蛾中的研究還是首次。本研究所獲得的一些結(jié)果為進一步探索在昆蟲免疫反應(yīng)中親免素A和親免素10起到的作用提供重要的信息。 提取斜紋夜蛾的血淋巴,反轉(zhuǎn)錄成cDNA,以cDNA為模版克隆得到兩個基因：SpliCypA和SpliCyplO。SpliCypA開放閱讀框ORF的大小為498bp,編碼165個aa,其理論分子量為17.62kDa。SpliCyp10開放閱讀框ORF的大小為490bp,編碼161個aa,其理論分子量為17.89kDa。斜紋夜蛾CypA的ORF編碼氨基酸的序列分別與大家鼠CypA、非洲爪蟾CypA、人CypA、家蠶CypA、粘蟲CypA的ORF編碼的氨基酸序列相比較,其同源性分別為74.4%、73.9%、76.8%、83.6%、和95.8%。得到了這些基本信息后,我們構(gòu)建了SpliCypA的原核表達載體pET32a-SpliCypA,并通過南京巴傲德生物科技有限公司制備得到了SpiCypA的抗體。在此基礎(chǔ)上,成功地構(gòu)建了真核表達載體pIZT/V5-His-SpliCypA, Western blot檢測到SpliCypA在四種昆蟲細胞(Bml2、Spli22、Sf9、High Five)中的均能表達,其融合蛋白的大小為21.29kDa。免疫熒光顯示CypA蛋白在High Five細胞的細胞質(zhì)中。用真核表達質(zhì)粒pIZT/V5-His-SpliCypA轉(zhuǎn)染High Five細胞后,利用RNA i技術(shù),沉默High Five細胞中的CypA,用制備得到的抗體進行檢測,我們發(fā)現(xiàn)實驗組pIZT/V5-His-CypA-siRNA-22和pIZT/V5-His-CypA-siRNA-37比對照組表達蛋白量均減弱。同時我們還構(gòu)建SpliCyp10真核表達載體pIZT/V5-His-SpliCyplO,其融合蛋白的分子量為21.75kDa。pIZT/V5-His-SpliCyp10轉(zhuǎn)染昆蟲細胞Sf9和Spil221,96h提取細胞的總蛋白,Western blot結(jié)果顯示該質(zhì)粒能在這兩種昆蟲細胞中有效表達,免疫熒光顯示Cyp10蛋白分布在細胞質(zhì)當中,靠近在細胞核的位置。
[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.
【學位授予單位】：云南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S433.4

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本文編號：1695364