家蚕脯氨酰寡肽酶家族的鉴定及BmAPH的体外表达与活性分析
发布时间：2018-07-23 15:38
【摘要】：脯氨酰寡肽酶家族(Prolyl oligopeptidase family)是丝氨酸蛋白酶超家族(Serine protease superfamily)中的一个家族,包括脯氨酰寡肽酶(Prolyl oligopeptidase,POP)、二肽基肽酶(Dipeptidyl peptidase,DDP)、乙酰肽水解酶(Acylpeptide hydrolase,APH)和谷氨酰内肽酶(Glutamyl endopeptidase,GEP)4个亚家族。在生物体内,这类酶可以调节生物活性肽及肽类激素的活性,参与很多重要的生理过程。在哺乳动物中与某些疾病息息相关,如健忘症、抑郁症、糖尿病和锥虫病等。脯氨酰寡肽酶与脂酶(Lipase)和酯酶(Esterase)类似,均有C-端α/β-水解酶催化结构域,N-端β-螺旋结构域可以调节活性部位的构象。脯氨酰寡肽酶在哺乳动物中研究较多,但在昆虫中研究较少。家蚕(Bombyx mori,B.mori),是由5000多年前的野桑蚕(Bombyx mandarina,B.mandarina)经人工饲养驯化而来。随着生物学的发展,家蚕作为鳞翅目的模式昆虫,已被广泛用于昆虫的比较基因组学和遗传学等方面的研究。近些年来,家蚕基因组序列公布后,越来越多的基因家族被鉴定,相关的基因功能相继被报道。但家蚕基因组中脯氨酰寡肽酶家族的信息及有关功能仍不清楚。本文基于家蚕基因组序列、预测的蛋白质库和EST数据,通过比较基因组学、生物信息学分析对家蚕脯氨酰寡肽酶家族进行鉴定、进化分析以及表达模式分析,并对家蚕乙酰肽水解酶基因(BmAPH)进行了克隆和功能分析,得到了如下主要结果:(1)基于家蚕的预测蛋白质库,利用hmmsearch检索,从家蚕基因组中鉴定出9个脯氨酰寡肽酶家族的候选基因,可以分为3个亚家族,其中APH、POP这两个亚家族均只有一个成员,DDP亚家族有7个成员。(2)基于候选基因的核苷酸序列设计引物,通过RT-PCR对脯氨酰寡肽酶家族的候选基因进行了表达模式分析。结果发现家蚕POP家族9个基因中8个有转录证据,Bm3和Bm5分别在幼虫期和蛹后期及成虫期高表达,Bm3在幼虫中肠中高表达,Bm5和Bm7在头、表皮、性腺中高表达。家蚕POP家族成员表达模式的差异为后续的功能研究提供了一定依据。(3)通过5’-RACE、3’-RACE以及RT-PCR从家蚕大造品系5龄3天整蚕材料中克隆获得BmAPH基因的全长序列(GenBank登陆号:KR094958)。BmAPH基因的cDNA全长2751 bp,开放阅读框(ORF)长2133 bp,5’-UTR长147 bp,3’-UTR长471 bp,可编码含710个氨基酸残基的蛋白质,预测分子量和等电点分别为78,481 Da和6.31。该基因由14个外显子和15个内含子组成,内含子的剪切边界含有保守的序列“GT-AG”。多序列比对,发现BmAPH含有乙酰肽水解酶的典型特征,如Ser566-Asp654-His686催化三联体、含有活性位点丝氨酸残基以及三个甘氨酸残基的一致序列(G-X-S566-X-G-G)和形成氧离子穴的保守基序(HGGP)。(4)构建原核表达载体BmAPH-pET28a,将BmAPH基因进行外源诱导表达。通过摸索诱导温度、诱导剂浓度以及诱导时间对外源蛋白表达形式的影响,从而得到了BmAPH蛋白以可溶形式表达的最佳条件。利用亲和层析的方法纯化含有组氨酸标签的重组蛋白,通过Western blotting以及Maldi-TOF-TOF质谱鉴定,确认纯化的蛋白确实是BmAPH基因的外源表达产物。(5)通过RT-PCR检测BmAPH基因的表达模式,结果发现BmAPH基因在被检测的9个组织和23个发育时间点均有表达,且几乎没有组织或时期表达特异性。以纯化的蛋白作为抗原免疫小鼠,制备多克隆抗体。以提取的大造5龄3天幼虫的总蛋白作为抗原,通过Western blotting验证,结果表明制备的多克隆抗体是特异性的,可用于后续的实验。对大造5龄3天幼虫头和中肠进行免疫组织化学分析,结果表明BmAPH存在于基膜中。基膜不仅对细胞、组织结构起支持作用,同时也是渗透性的障碍,可调节分子和细胞的运动,一定程度上可使细胞或组织免受损伤。(6)纯化蛋白的酶活测定表明,BmAPH具有乙酰肽水解酶活性。通过测定不同反应温度或同一温度下不同pH对酶活性的影响,发现BmAPH活性易受温度和pH的影响,最适反应温度和最适pH值分别为50℃和7.7。分析三种有机磷杀虫剂(毒死蜱、马拉硫磷和辛硫磷)对BmAPH活性的影响,发现它们均能抑制BmAPH的活性。马拉硫磷和辛硫磷对BmAPH活性的抑制中浓度IC50分别为7.62 mg/L和7.90 mg/L,相对而言,毒死蜱对BmAPH活性的抑制程度更高,IC50为1.60 mg/L。同时,测定用有机磷杀虫剂处理家蚕后整蚕和中肠中APH的活性。与对照相比,结果表明分别用杀虫剂处理家蚕后整蚕或中肠中APH的活性显著地降低了。综上所述,从家蚕基因组中鉴定出9个脯氨酰寡肽酶家族候选基因,与其他物种脯氨酰寡肽酶家族基因进行了比较分析,并从分子水平检测了该家族基因在家蚕不同组织以及不同发育时期的表达模式,这为研究脯氨酰寡肽酶家族基因在家蚕及其他昆虫中的潜在功能或进化关系提供了一定依据。另外,对BmAPH进行了克隆、表达模式、组织定位和酶活性分析,为研究其他昆虫中乙酰肽水解酶的功能提供了有用信息。重要的是,本研究结果表明有机磷杀虫剂能抑制BmAPH的活性,说明BmAPH是有机磷杀虫剂的敏感靶标。
[Abstract]:The prolyl oligopeptidase family (Prolyl oligopeptidase family) is a family in the serine protease superfamily (Serine protease superfamily), including prolyl oligopeptidase (Prolyl oligopeptidase, POP), two peptidyl peptidase (Dipeptidyl peptidase, DDP), acetyl peptide hydrolase and glutamyl endopeptidase. YL endopeptidase, GEP) 4 subfamilies. In vivo, these enzymes can regulate the activity of bioactive peptides and peptide hormones and participate in many important physiological processes. In mammals, it is closely related to some diseases, such as amnesia, depression, diabetes and trypanosomiasis. Prolyl oligopeptidase, lipase (Lipase) and esterase (Esterase) class. It seems that the C- terminal alpha / beta hydrolase catalyzes the domain, and the N- end beta spiral domain can regulate the conformation of the active site. The prolyl oligopeptidase is studied more in mammals, but less in the insect. The Bombyx mori (B.mori) of the silkworm (Bombyx Mandarina, B.mandarina) was artificially reared and acclimated by the silkworm (Bombyx Mandarina, B.mandarina) 5000 years ago. With the development of biology, silkworm, as a model insect of Lepidoptera, has been widely used in the research of comparative genomics and genetics of insects. In recent years, more and more gene families have been identified and related gene functions have been reported after the publication of the silkworm genome sequence. But the prolyyl oligopeptidase family in the silkworm genome The information and related functions are still not clear. Based on the sequence of the silkworm genome, the predicted protein library and the EST data, the prolyyl oligopeptidase family of the silkworm was identified, the evolution analysis and the expression pattern analysis were analyzed by comparative genomics and bioinformatics analysis, and the clone and work of the BmAPH gene of the family silkworm acetyl peptide hydrolase (BmAPH) were cloned and worked. The main results are as follows: (1) based on the prediction of the protein library of the silkworm, the candidate genes of 9 prolyl oligopeptidase families in the silkworm genome are identified by hmmsearch retrieval, which can be divided into 3 subfamilies, of which there are only one member of the two subfamilies of APH, POP and 7 members of the DDP subfamily. (2) based on the candidate genes. The nucleotide sequence was designed and the expression pattern of the prolyl oligopeptidase family was analyzed by RT-PCR. The results showed that 8 of the 9 genes of the POP family were transcriptional evidences, Bm3 and Bm5 were highly expressed in the larval stage and late pupal stage and adult stage respectively. Bm3 was highly expressed in the larva, and Bm5 and Bm7 were in the head, epidermis and gonads. High expression. The difference in expression patterns of the POP family members provided a basis for subsequent functional studies. (3) the full length of the full-length sequence of the BmAPH gene (GenBank landing number: KR094958).BmAPH gene was obtained from the 5 '-RACE, 3' -RACE and RT-PCR, and the open reading frame (ORF) was obtained by cloning the whole long sequence of the BmAPH gene from the 5 instar 3 day silkworm material of the Bombyx mori. ) long 2133 BP, 5 '-UTR long 147 BP, 3' -UTR long 471 BP, can encode a protein containing 710 amino acid residues. The molecular weight and isoelectric point are 78481 Da and 6.31., respectively, and the gene is composed of 14 exons and 15 introns. The shear boundary of the introns contains a conservative sequence "GT-AG". The typical characteristics of the hydrolytic enzyme, such as Ser566-Asp654-His686 catalyzing three body, containing the active site serine residues and the consistent sequence of three glycine residues (G-X-S566-X-G-G) and the formation of the conservative order of the oxygen ions (HGGP). (4) the prokaryotic expression vector, BmAPH-pET28a, was constructed to induce the expression of the BmAPH gene. The effect of inducer concentration and induction time on the expression of exogenous protein was obtained, and the optimum conditions for the expression of BmAPH protein in soluble form were obtained. The recombinant protein containing histidine label was purified by affinity chromatography. The purified protein was confirmed to be a BmAPH gene by Western blotting and Maldi-TOF-TOF mass spectrometry. (5) the expression pattern of BmAPH gene was detected by RT-PCR. The results showed that the BmAPH gene was expressed in 9 tissues and 23 developing time points, and almost no tissue or time expression. The purified protein was used as antigen to immunize mice and to produce polyclonal antibody. 5 instar 3 day larvae were extracted. The total protein, as antigen, was verified by Western blotting. The results showed that the polyclonal antibody prepared was specific and could be used for subsequent experiments. Immunohistochemical analysis of the head and midgut of the 5 instar 3 days larvae showed that BmAPH existed in the basement membrane. The barrier of permeability can regulate the movement of molecules and cells to some extent. (6) enzyme activity determination of purified protein shows that BmAPH has the activity of acetylpeptide hydrolase. By determining the effect of different reaction temperature or different pH on the enzyme activity at the same temperature, it is found that the activity of BmAPH is susceptible to temperature and pH. The reaction temperature and the optimum pH value were 50 C and 7.7. respectively to analyze the effects of three organophosphorus insecticides (chlorpyrifos, malathion and phoxim) on the activity of BmAPH, and they were found to inhibit the activity of BmAPH. The concentration of malathion and phoxim in the inhibition of BmAPH activity was 7.62 mg/L and 7.90 mg/L, respectively. The inhibition of H activity was higher, IC50 was 1.60 mg/L. and the activity of APH in silkworm and midgut of silkworm was measured with organophosphorus insecticides. Compared with the control, the results showed that the activity of APH in the silkworm and midgut of the silkworm was significantly reduced by the insecticide treatment. In summary, 9 prolyl Oligopeptides were identified from the genome of the silkworm. The enzyme family candidate genes were compared with the prolyl oligopeptidase family genes in other species, and the expression patterns of the family gene in different tissues and different developmental stages of the family silkworm were detected at the molecular level, which provided the potential function or evolutionary relationship of the prolyl oligopeptidase family gene in the silkworm and his insects. In addition, the cloning, expression pattern, tissue localization and enzyme activity analysis of BmAPH provide useful information for the study of the function of acetylpeptide hydrolase in other insects. It is important that the results of this study show that organophosphorus insecticides can inhibit the activity of BmAPH, and that BmAPH is a sensitive target for organophosphorus insecticides.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q55;Q78
，
本文编号：2139860
[Abstract]:The prolyl oligopeptidase family (Prolyl oligopeptidase family) is a family in the serine protease superfamily (Serine protease superfamily), including prolyl oligopeptidase (Prolyl oligopeptidase, POP), two peptidyl peptidase (Dipeptidyl peptidase, DDP), acetyl peptide hydrolase and glutamyl endopeptidase. YL endopeptidase, GEP) 4 subfamilies. In vivo, these enzymes can regulate the activity of bioactive peptides and peptide hormones and participate in many important physiological processes. In mammals, it is closely related to some diseases, such as amnesia, depression, diabetes and trypanosomiasis. Prolyl oligopeptidase, lipase (Lipase) and esterase (Esterase) class. It seems that the C- terminal alpha / beta hydrolase catalyzes the domain, and the N- end beta spiral domain can regulate the conformation of the active site. The prolyl oligopeptidase is studied more in mammals, but less in the insect. The Bombyx mori (B.mori) of the silkworm (Bombyx Mandarina, B.mandarina) was artificially reared and acclimated by the silkworm (Bombyx Mandarina, B.mandarina) 5000 years ago. With the development of biology, silkworm, as a model insect of Lepidoptera, has been widely used in the research of comparative genomics and genetics of insects. In recent years, more and more gene families have been identified and related gene functions have been reported after the publication of the silkworm genome sequence. But the prolyyl oligopeptidase family in the silkworm genome The information and related functions are still not clear. Based on the sequence of the silkworm genome, the predicted protein library and the EST data, the prolyyl oligopeptidase family of the silkworm was identified, the evolution analysis and the expression pattern analysis were analyzed by comparative genomics and bioinformatics analysis, and the clone and work of the BmAPH gene of the family silkworm acetyl peptide hydrolase (BmAPH) were cloned and worked. The main results are as follows: (1) based on the prediction of the protein library of the silkworm, the candidate genes of 9 prolyl oligopeptidase families in the silkworm genome are identified by hmmsearch retrieval, which can be divided into 3 subfamilies, of which there are only one member of the two subfamilies of APH, POP and 7 members of the DDP subfamily. (2) based on the candidate genes. The nucleotide sequence was designed and the expression pattern of the prolyl oligopeptidase family was analyzed by RT-PCR. The results showed that 8 of the 9 genes of the POP family were transcriptional evidences, Bm3 and Bm5 were highly expressed in the larval stage and late pupal stage and adult stage respectively. Bm3 was highly expressed in the larva, and Bm5 and Bm7 were in the head, epidermis and gonads. High expression. The difference in expression patterns of the POP family members provided a basis for subsequent functional studies. (3) the full length of the full-length sequence of the BmAPH gene (GenBank landing number: KR094958).BmAPH gene was obtained from the 5 '-RACE, 3' -RACE and RT-PCR, and the open reading frame (ORF) was obtained by cloning the whole long sequence of the BmAPH gene from the 5 instar 3 day silkworm material of the Bombyx mori. ) long 2133 BP, 5 '-UTR long 147 BP, 3' -UTR long 471 BP, can encode a protein containing 710 amino acid residues. The molecular weight and isoelectric point are 78481 Da and 6.31., respectively, and the gene is composed of 14 exons and 15 introns. The shear boundary of the introns contains a conservative sequence "GT-AG". The typical characteristics of the hydrolytic enzyme, such as Ser566-Asp654-His686 catalyzing three body, containing the active site serine residues and the consistent sequence of three glycine residues (G-X-S566-X-G-G) and the formation of the conservative order of the oxygen ions (HGGP). (4) the prokaryotic expression vector, BmAPH-pET28a, was constructed to induce the expression of the BmAPH gene. The effect of inducer concentration and induction time on the expression of exogenous protein was obtained, and the optimum conditions for the expression of BmAPH protein in soluble form were obtained. The recombinant protein containing histidine label was purified by affinity chromatography. The purified protein was confirmed to be a BmAPH gene by Western blotting and Maldi-TOF-TOF mass spectrometry. (5) the expression pattern of BmAPH gene was detected by RT-PCR. The results showed that the BmAPH gene was expressed in 9 tissues and 23 developing time points, and almost no tissue or time expression. The purified protein was used as antigen to immunize mice and to produce polyclonal antibody. 5 instar 3 day larvae were extracted. The total protein, as antigen, was verified by Western blotting. The results showed that the polyclonal antibody prepared was specific and could be used for subsequent experiments. Immunohistochemical analysis of the head and midgut of the 5 instar 3 days larvae showed that BmAPH existed in the basement membrane. The barrier of permeability can regulate the movement of molecules and cells to some extent. (6) enzyme activity determination of purified protein shows that BmAPH has the activity of acetylpeptide hydrolase. By determining the effect of different reaction temperature or different pH on the enzyme activity at the same temperature, it is found that the activity of BmAPH is susceptible to temperature and pH. The reaction temperature and the optimum pH value were 50 C and 7.7. respectively to analyze the effects of three organophosphorus insecticides (chlorpyrifos, malathion and phoxim) on the activity of BmAPH, and they were found to inhibit the activity of BmAPH. The concentration of malathion and phoxim in the inhibition of BmAPH activity was 7.62 mg/L and 7.90 mg/L, respectively. The inhibition of H activity was higher, IC50 was 1.60 mg/L. and the activity of APH in silkworm and midgut of silkworm was measured with organophosphorus insecticides. Compared with the control, the results showed that the activity of APH in the silkworm and midgut of the silkworm was significantly reduced by the insecticide treatment. In summary, 9 prolyl Oligopeptides were identified from the genome of the silkworm. The enzyme family candidate genes were compared with the prolyl oligopeptidase family genes in other species, and the expression patterns of the family gene in different tissues and different developmental stages of the family silkworm were detected at the molecular level, which provided the potential function or evolutionary relationship of the prolyl oligopeptidase family gene in the silkworm and his insects. In addition, the cloning, expression pattern, tissue localization and enzyme activity analysis of BmAPH provide useful information for the study of the function of acetylpeptide hydrolase in other insects. It is important that the results of this study show that organophosphorus insecticides can inhibit the activity of BmAPH, and that BmAPH is a sensitive target for organophosphorus insecticides.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q55;Q78
，
本文编号：2139860
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