家蚕类胰岛素相关肽结合蛋白2的功能分析及其互作蛋白的鉴定
发布时间：2018-11-19 18:57
【摘要】：家蚕是鳞翅目昆虫的模式生物,也是重要的遗传研究模型。家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,Bm CPV)是家蚕主要病毒病原之一,可以引起家蚕群体内中肠型脓病的发生。迄今为止,国内外有关家蚕对BmCPV的防御机制的研究报道较少,养蚕生产上对中肠型脓病的防治也仅是以预防为主。不同家蚕品种对BmCPV感染的抵抗性有一定的差异,但尚未培育出抗BmCPV的家蚕品种。为了从分子水平上探索家蚕抵抗BmCPV感染的防御机制和不同家蚕品种对BmCPV的免疫调控机理,我们前期通过高通量测序技术分析筛选了家蚕品种4008(BmCPV易感)和P50(BmCPV抗性)在感染BmCPV后的差异表达基因,发现家蚕中肠的类胰岛素相关肽结合蛋白2(Bombyx mori insulin-related peptide-binding protein,BmIBP2)基因的表达水平明显上调,且在抗性品种中的上调表达倍数高于易感品种,生物信息学分析推测IBP基因与细胞凋亡及免疫防御有关,因此推测BmIBP2参与了家蚕抵抗BmCPV感染的防御机制。本研究成功表达了BmIBP2的重组蛋白rBmIBP2,研究该蛋白对家蚕细胞增殖的影响,并采用RNA干涉及过表达技术研究了BmIBP2基因的功能,进一步鉴定获得一个BmIBP2的互作蛋白。得到的主要实验结果如下:1.BmIBP2基因在Bm CPV和其它几种病原侵染家蚕中肠中的差异表达通过实时荧光定量PCR(qRT-PCR)技术检测了家蚕感染质型多角体病毒(BmCPV)、核型多角体病毒(BmNPV)、二分浓核病毒(BmBDV)、球孢白僵菌(Beauveria bassina)、家蚕微孢子虫(Nosema bombycis)后不同时间点中肠组织中BmIBP2的表达情况,结果发现BmIBP2基因不仅在BmCPV感染的家蚕中肠上调表达,在其他病原感染的家蚕中肠,也发生一定的差异表达,说明该基因参与了家蚕对病原感染的应答与防御过程,并且具有重要的免疫防御功能。2.BmIBP2蛋白对家蚕培养细胞增殖的影响首先通过原核表达系统对BmIBP2基因进行重组表达,对表达的重组蛋白进行纯化复性后添加入家蚕BmN细胞培养基中,通过MTT法检测该纯化蛋白对BmN细胞增殖的影响,并检测了蜕皮激素20E对BmIBP2基因的调控作用。结果表明,纯化的重组蛋白rBm IBP2对家蚕BmN细胞的增殖具有一定的抑制作用,并且20E能够促进家蚕细胞中BmIBP2的表达。进一步通过RNAi和基因过表达技术探索了BmIBP2基因的功能。采用化学合成法合成了BmIBP2基因的si RNA及negative control siRNA,转染家蚕BmN细胞,检测其对细胞增殖的影响,同时检测BmIBP2基因及糖代谢相关呼吸酶基因的表达变化情况。RNAi实验结果显示,BmIBP2基因被干扰沉默后,与对照组相比,BmIBP2基因呈现下调表达,家蚕BmN细胞增殖速度加快,同时检测到糖代谢相关呼吸酶基因呈现上调表达。同时,我们构建了转基因表达载体pIZT-IBP2对BmIBP2基因进行过表达,实验结果显示,与对照组相比家蚕BmN细胞增殖速度减慢,并且糖代谢呼吸酶相关基因呈现下调表达。3.BmIBP2互作蛋白的筛选与鉴定利用Bac-to-Bac表达系统将BmIBP2和EGFP在家蚕细胞中进行融合表达,其中EGFP基因作为报告基因,经同源重组获得vBmEGFP-IBP2重组杆状病毒穿梭质粒。转染家蚕细胞并用SDS-PAGE和Western-blot方法检测重组蛋白的表达,结果发现重组蛋白成功表达,大小约为55kDa。随后收集蛋白并利用EGFP作为抗原决定簇通过免疫共沉淀技术筛选与BmIBP2蛋白相互作用的宿主细胞蛋白。通过免疫共沉淀实验,我们筛选到一个与BmIBP2有相互作用的蛋白,经质谱鉴定为家蚕ATP合成酶(Bombyx mori ATP synthase)。经酵母双杂交实验验证,家蚕类胰岛素相关肽结合蛋白2与家蚕ATP合成酶之间的确存在相互作用。上述研究结果不仅可为深入了解BmIBP2基因参与家蚕抵抗BmCPV感染的途径及其作用机理提供了有力依据,而且为进一步揭示BmIBP2的生物学功能及家蚕抗BmCPV的分子机制奠定了良好的基础和依据。
[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.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S884.51
本文编号：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.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S884.51
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