家蠶類胰島素相關(guān)肽結(jié)合蛋白2的功能分析及其互作蛋白的鑒定
發(fā)布時間：2018-11-19 18:57
【摘要】：家蠶是鱗翅目昆蟲的模式生物,也是重要的遺傳研究模型。家蠶質(zhì)型多角體病毒(Bombyx mori cytoplasmic polyhedrosis virus,Bm CPV)是家蠶主要病毒病原之一,可以引起家蠶群體內(nèi)中腸型膿病的發(fā)生。迄今為止,國內(nèi)外有關(guān)家蠶對BmCPV的防御機(jī)制的研究報道較少,養(yǎng)蠶生產(chǎn)上對中腸型膿病的防治也僅是以預(yù)防為主。不同家蠶品種對BmCPV感染的抵抗性有一定的差異,但尚未培育出抗BmCPV的家蠶品種。為了從分子水平上探索家蠶抵抗BmCPV感染的防御機(jī)制和不同家蠶品種對BmCPV的免疫調(diào)控機(jī)理,我們前期通過高通量測序技術(shù)分析篩選了家蠶品種4008(BmCPV易感)和P50(BmCPV抗性)在感染BmCPV后的差異表達(dá)基因,發(fā)現(xiàn)家蠶中腸的類胰島素相關(guān)肽結(jié)合蛋白2(Bombyx mori insulin-related peptide-binding protein,BmIBP2)基因的表達(dá)水平明顯上調(diào),且在抗性品種中的上調(diào)表達(dá)倍數(shù)高于易感品種,生物信息學(xué)分析推測IBP基因與細(xì)胞凋亡及免疫防御有關(guān),因此推測BmIBP2參與了家蠶抵抗BmCPV感染的防御機(jī)制。本研究成功表達(dá)了BmIBP2的重組蛋白rBmIBP2,研究該蛋白對家蠶細(xì)胞增殖的影響,并采用RNA干涉及過表達(dá)技術(shù)研究了BmIBP2基因的功能,進(jìn)一步鑒定獲得一個BmIBP2的互作蛋白。得到的主要實(shí)驗(yàn)結(jié)果如下:1.BmIBP2基因在Bm CPV和其它幾種病原侵染家蠶中腸中的差異表達(dá)通過實(shí)時熒光定量PCR(qRT-PCR)技術(shù)檢測了家蠶感染質(zhì)型多角體病毒(BmCPV)、核型多角體病毒(BmNPV)、二分濃核病毒(BmBDV)、球孢白僵菌(Beauveria bassina)、家蠶微孢子蟲(Nosema bombycis)后不同時間點(diǎn)中腸組織中BmIBP2的表達(dá)情況,結(jié)果發(fā)現(xiàn)BmIBP2基因不僅在BmCPV感染的家蠶中腸上調(diào)表達(dá),在其他病原感染的家蠶中腸,也發(fā)生一定的差異表達(dá),說明該基因參與了家蠶對病原感染的應(yīng)答與防御過程,并且具有重要的免疫防御功能。2.BmIBP2蛋白對家蠶培養(yǎng)細(xì)胞增殖的影響首先通過原核表達(dá)系統(tǒng)對BmIBP2基因進(jìn)行重組表達(dá),對表達(dá)的重組蛋白進(jìn)行純化復(fù)性后添加入家蠶BmN細(xì)胞培養(yǎng)基中,通過MTT法檢測該純化蛋白對BmN細(xì)胞增殖的影響,并檢測了蛻皮激素20E對BmIBP2基因的調(diào)控作用。結(jié)果表明,純化的重組蛋白rBm IBP2對家蠶BmN細(xì)胞的增殖具有一定的抑制作用,并且20E能夠促進(jìn)家蠶細(xì)胞中BmIBP2的表達(dá)。進(jìn)一步通過RNAi和基因過表達(dá)技術(shù)探索了BmIBP2基因的功能。采用化學(xué)合成法合成了BmIBP2基因的si RNA及negative control siRNA,轉(zhuǎn)染家蠶BmN細(xì)胞,檢測其對細(xì)胞增殖的影響,同時檢測BmIBP2基因及糖代謝相關(guān)呼吸酶基因的表達(dá)變化情況。RNAi實(shí)驗(yàn)結(jié)果顯示,BmIBP2基因被干擾沉默后,與對照組相比,BmIBP2基因呈現(xiàn)下調(diào)表達(dá),家蠶BmN細(xì)胞增殖速度加快,同時檢測到糖代謝相關(guān)呼吸酶基因呈現(xiàn)上調(diào)表達(dá)。同時,我們構(gòu)建了轉(zhuǎn)基因表達(dá)載體pIZT-IBP2對BmIBP2基因進(jìn)行過表達(dá),實(shí)驗(yàn)結(jié)果顯示,與對照組相比家蠶BmN細(xì)胞增殖速度減慢,并且糖代謝呼吸酶相關(guān)基因呈現(xiàn)下調(diào)表達(dá)。3.BmIBP2互作蛋白的篩選與鑒定利用Bac-to-Bac表達(dá)系統(tǒng)將BmIBP2和EGFP在家蠶細(xì)胞中進(jìn)行融合表達(dá),其中EGFP基因作為報告基因,經(jīng)同源重組獲得vBmEGFP-IBP2重組桿狀病毒穿梭質(zhì)粒。轉(zhuǎn)染家蠶細(xì)胞并用SDS-PAGE和Western-blot方法檢測重組蛋白的表達(dá),結(jié)果發(fā)現(xiàn)重組蛋白成功表達(dá),大小約為55kDa。隨后收集蛋白并利用EGFP作為抗原決定簇通過免疫共沉淀技術(shù)篩選與BmIBP2蛋白相互作用的宿主細(xì)胞蛋白。通過免疫共沉淀實(shí)驗(yàn),我們篩選到一個與BmIBP2有相互作用的蛋白,經(jīng)質(zhì)譜鑒定為家蠶ATP合成酶(Bombyx mori ATP synthase)。經(jīng)酵母雙雜交實(shí)驗(yàn)驗(yàn)證,家蠶類胰島素相關(guān)肽結(jié)合蛋白2與家蠶ATP合成酶之間的確存在相互作用。上述研究結(jié)果不僅可為深入了解BmIBP2基因參與家蠶抵抗BmCPV感染的途徑及其作用機(jī)理提供了有力依據(jù),而且為進(jìn)一步揭示BmIBP2的生物學(xué)功能及家蠶抗BmCPV的分子機(jī)制奠定了良好的基礎(chǔ)和依據(jù)。
[Abstract]:The silkworm is a model organism of lepidopteran insects, and is also an important genetic research model. Bom CPV (Bombyx mori) is one of the major virus pathogens in the silkworm, which can cause the occurrence of intestinal type empyema in the silkworm population. So far, there are few studies on the defense mechanism of BmCPV at home and abroad. The resistance of different silkworm varieties to BmCPV infection is different, but the silkworm variety with anti-BmCPV has not been cultivated. In order to study the defense mechanism of BmCPV infection and the immune regulation mechanism of different silkworm varieties to BmCPV from the molecular level, the differential expression genes of the silkworm variety 4008 (BmCPV susceptibility) and P50 (BmCPV resistance) after the infection of BmCPV were analyzed by high-throughput sequencing technology. It was found that the level of the expression of the related peptide-related peptide-binding protein 2 (BmIBP2) gene of the intestine in the silkworm was up-regulated, and the up-regulation expression in the resistant variety was higher than that of the susceptible variety, and the bioinformatics analysis suggested that the IBP gene was related to the cell apoptosis and the immune defense. Therefore, it is assumed that BmIBP2 is involved in the defense mechanism of the silkworm against the BmCPV infection. The recombinant protein rBmIBP2 of BmIBP2 was successfully expressed in the study, and the effect of the protein on the proliferation of the silkworm cells was studied. The function of the BmIBP2 gene was studied by means of RNA interference and over-expression. The main experimental results are as follows: 1. The differential expression of BmIBP2 gene in the intestine of Bm CPV and several other pathogens in the intestine is detected by the real-time fluorescence quantitative PCR (qRT-PCR) technology. The nucleopolyhedrosis virus (BmCPV), the nuclear polyhedrosis virus (BmNPV) and the bipartite concentrated nuclear virus (BmBDV) are detected by the real-time fluorescence quantitative PCR (qRT-PCR) technology. The expression of BmIB2 in intestinal tissue at different time points of Beauveria bassiana and Nosema bombycis was observed. The results showed that the BmIBP2 gene not only increased the expression of BmIB2 in the silkworm of BmCPV infection, but also expressed a certain difference in the intestine in the silkworm of other pathogenic infection. the gene is involved in the response and defense process of the silkworm to the pathogenic infection, and has an important immune defense function. The expression recombinant protein was purified and renaturation, then added into the BmN cell culture medium of the silkworm, and the effect of the purified protein on the proliferation of the BmN cells was detected by the MTT method, and the regulation effect of the ecdysone 20E on the BmIBP2 gene was detected. The results showed that the purified recombinant protein rBmIBP2 has a certain inhibitory effect on the proliferation of BmN cells in the silkworm, and 20E can promote the expression of BmIBP2 in the silkworm cell. The function of the BmIBP2 gene is further explored by the RNAi and the gene overexpression technology. The si RNA and the native control siRNA of the BmIBP2 gene were synthesized by the chemical synthesis method, and the BmN cells were transfected into the silkworm BmN cells, and the effect of the BmIBP2 gene and the sugar metabolism related respiratory enzyme gene was detected. The results of RNAi showed that, after the BmIBP2 gene was disturbed and silent, the BmIBP2 gene was down-regulated compared with the control group, and the proliferation rate of the BmN cells in the silkworm was increased, and the expression of the related respiratory enzyme gene of the sugar metabolism was detected up-regulated. in that meantime, we construct the transgenic expression vector pIZT-IBP2 to overexpress the BmIBP2 gene, and the result shows that the proliferation rate of the BmN cell in the silkworm is slowed down compared with the control group, and expressing the BmIBP2 and EGFP in the silkworm cell by using the Bac-to-Bac expression system, wherein the EGFP gene is used as a reporter gene, and the vBmEGFP-IBP2 recombinant baculovirus shuttle plasmid is obtained through homologous recombination. The expression of recombinant protein was detected by SDS-PAGE and Western-blot, and the expression of recombinant protein was found to be about 55kDa. The protein was then collected and the host cell protein interacting with the BmIBP2 protein was screened by the immunoprecipitation technique using EGFP as an antigenic determinant. Through the immunoprecipitation experiment, we screened a protein that interacts with BmIBP2, and was identified by mass spectrometry as the silkworm ATP synthase. It is verified by yeast two-hybrid experiment that there is a real interaction between the related peptide binding protein 2 and the silkworm ATP synthase in the silkworm. The results of the above research not only provide a powerful basis for the in-depth understanding of the BmIBP2 gene involved in the BmCPV infection, but also provide a good basis for further revealing the biological function of BmIBP2 and the molecular mechanism of the silkworm anti-BmCPV.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S884.51
本文編號：2343151
[Abstract]:The silkworm is a model organism of lepidopteran insects, and is also an important genetic research model. Bom CPV (Bombyx mori) is one of the major virus pathogens in the silkworm, which can cause the occurrence of intestinal type empyema in the silkworm population. So far, there are few studies on the defense mechanism of BmCPV at home and abroad. The resistance of different silkworm varieties to BmCPV infection is different, but the silkworm variety with anti-BmCPV has not been cultivated. In order to study the defense mechanism of BmCPV infection and the immune regulation mechanism of different silkworm varieties to BmCPV from the molecular level, the differential expression genes of the silkworm variety 4008 (BmCPV susceptibility) and P50 (BmCPV resistance) after the infection of BmCPV were analyzed by high-throughput sequencing technology. It was found that the level of the expression of the related peptide-related peptide-binding protein 2 (BmIBP2) gene of the intestine in the silkworm was up-regulated, and the up-regulation expression in the resistant variety was higher than that of the susceptible variety, and the bioinformatics analysis suggested that the IBP gene was related to the cell apoptosis and the immune defense. Therefore, it is assumed that BmIBP2 is involved in the defense mechanism of the silkworm against the BmCPV infection. The recombinant protein rBmIBP2 of BmIBP2 was successfully expressed in the study, and the effect of the protein on the proliferation of the silkworm cells was studied. The function of the BmIBP2 gene was studied by means of RNA interference and over-expression. The main experimental results are as follows: 1. The differential expression of BmIBP2 gene in the intestine of Bm CPV and several other pathogens in the intestine is detected by the real-time fluorescence quantitative PCR (qRT-PCR) technology. The nucleopolyhedrosis virus (BmCPV), the nuclear polyhedrosis virus (BmNPV) and the bipartite concentrated nuclear virus (BmBDV) are detected by the real-time fluorescence quantitative PCR (qRT-PCR) technology. The expression of BmIB2 in intestinal tissue at different time points of Beauveria bassiana and Nosema bombycis was observed. The results showed that the BmIBP2 gene not only increased the expression of BmIB2 in the silkworm of BmCPV infection, but also expressed a certain difference in the intestine in the silkworm of other pathogenic infection. the gene is involved in the response and defense process of the silkworm to the pathogenic infection, and has an important immune defense function. The expression recombinant protein was purified and renaturation, then added into the BmN cell culture medium of the silkworm, and the effect of the purified protein on the proliferation of the BmN cells was detected by the MTT method, and the regulation effect of the ecdysone 20E on the BmIBP2 gene was detected. The results showed that the purified recombinant protein rBmIBP2 has a certain inhibitory effect on the proliferation of BmN cells in the silkworm, and 20E can promote the expression of BmIBP2 in the silkworm cell. The function of the BmIBP2 gene is further explored by the RNAi and the gene overexpression technology. The si RNA and the native control siRNA of the BmIBP2 gene were synthesized by the chemical synthesis method, and the BmN cells were transfected into the silkworm BmN cells, and the effect of the BmIBP2 gene and the sugar metabolism related respiratory enzyme gene was detected. The results of RNAi showed that, after the BmIBP2 gene was disturbed and silent, the BmIBP2 gene was down-regulated compared with the control group, and the proliferation rate of the BmN cells in the silkworm was increased, and the expression of the related respiratory enzyme gene of the sugar metabolism was detected up-regulated. in that meantime, we construct the transgenic expression vector pIZT-IBP2 to overexpress the BmIBP2 gene, and the result shows that the proliferation rate of the BmN cell in the silkworm is slowed down compared with the control group, and expressing the BmIBP2 and EGFP in the silkworm cell by using the Bac-to-Bac expression system, wherein the EGFP gene is used as a reporter gene, and the vBmEGFP-IBP2 recombinant baculovirus shuttle plasmid is obtained through homologous recombination. The expression of recombinant protein was detected by SDS-PAGE and Western-blot, and the expression of recombinant protein was found to be about 55kDa. The protein was then collected and the host cell protein interacting with the BmIBP2 protein was screened by the immunoprecipitation technique using EGFP as an antigenic determinant. Through the immunoprecipitation experiment, we screened a protein that interacts with BmIBP2, and was identified by mass spectrometry as the silkworm ATP synthase. It is verified by yeast two-hybrid experiment that there is a real interaction between the related peptide binding protein 2 and the silkworm ATP synthase in the silkworm. The results of the above research not only provide a powerful basis for the in-depth understanding of the BmIBP2 gene involved in the BmCPV infection, but also provide a good basis for further revealing the biological function of BmIBP2 and the molecular mechanism of the silkworm anti-BmCPV.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S884.51
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