家蠶脯氨酰寡肽酶家族的鑒定及BmAPH的體外表達與活性分析
發(fā)布時間：2018-07-23 15:38
【摘要】：脯氨酰寡肽酶家族(Prolyl oligopeptidase family)是絲氨酸蛋白酶超家族(Serine protease superfamily)中的一個家族,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)和谷氨酰內(nèi)肽酶(Glutamyl endopeptidase,GEP)4個亞家族。在生物體內(nèi),這類酶可以調(diào)節(jié)生物活性肽及肽類激素的活性,參與很多重要的生理過程。在哺乳動物中與某些疾病息息相關,如健忘癥、抑郁癥、糖尿病和錐蟲病等。脯氨酰寡肽酶與脂酶(Lipase)和酯酶(Esterase)類似,均有C-端α/β-水解酶催化結構域,N-端β-螺旋結構域可以調(diào)節(jié)活性部位的構象。脯氨酰寡肽酶在哺乳動物中研究較多,但在昆蟲中研究較少。家蠶(Bombyx mori,B.mori),是由5000多年前的野桑蠶(Bombyx mandarina,B.mandarina)經(jīng)人工飼養(yǎng)馴化而來。隨著生物學的發(fā)展,家蠶作為鱗翅目的模式昆蟲,已被廣泛用于昆蟲的比較基因組學和遺傳學等方面的研究。近些年來,家蠶基因組序列公布后,越來越多的基因家族被鑒定,相關的基因功能相繼被報道。但家蠶基因組中脯氨酰寡肽酶家族的信息及有關功能仍不清楚。本文基于家蠶基因組序列、預測的蛋白質(zhì)庫和EST數(shù)據(jù),通過比較基因組學、生物信息學分析對家蠶脯氨酰寡肽酶家族進行鑒定、進化分析以及表達模式分析,并對家蠶乙酰肽水解酶基因(BmAPH)進行了克隆和功能分析,得到了如下主要結果:(1)基于家蠶的預測蛋白質(zhì)庫,利用hmmsearch檢索,從家蠶基因組中鑒定出9個脯氨酰寡肽酶家族的候選基因,可以分為3個亞家族,其中APH、POP這兩個亞家族均只有一個成員,DDP亞家族有7個成員。(2)基于候選基因的核苷酸序列設計引物,通過RT-PCR對脯氨酰寡肽酶家族的候選基因進行了表達模式分析。結果發(fā)現(xiàn)家蠶POP家族9個基因中8個有轉錄證據(jù),Bm3和Bm5分別在幼蟲期和蛹后期及成蟲期高表達,Bm3在幼蟲中腸中高表達,Bm5和Bm7在頭、表皮、性腺中高表達。家蠶POP家族成員表達模式的差異為后續(xù)的功能研究提供了一定依據(jù)。(3)通過5’-RACE、3’-RACE以及RT-PCR從家蠶大造品系5齡3天整蠶材料中克隆獲得BmAPH基因的全長序列(GenBank登陸號:KR094958)。BmAPH基因的cDNA全長2751 bp,開放閱讀框(ORF)長2133 bp,5’-UTR長147 bp,3’-UTR長471 bp,可編碼含710個氨基酸殘基的蛋白質(zhì),預測分子量和等電點分別為78,481 Da和6.31。該基因由14個外顯子和15個內(nèi)含子組成,內(nèi)含子的剪切邊界含有保守的序列“GT-AG”。多序列比對,發(fā)現(xiàn)BmAPH含有乙酰肽水解酶的典型特征,如Ser566-Asp654-His686催化三聯(lián)體、含有活性位點絲氨酸殘基以及三個甘氨酸殘基的一致序列(G-X-S566-X-G-G)和形成氧離子穴的保守基序(HGGP)。(4)構建原核表達載體BmAPH-pET28a,將BmAPH基因進行外源誘導表達。通過摸索誘導溫度、誘導劑濃度以及誘導時間對外源蛋白表達形式的影響,從而得到了BmAPH蛋白以可溶形式表達的最佳條件。利用親和層析的方法純化含有組氨酸標簽的重組蛋白,通過Western blotting以及Maldi-TOF-TOF質(zhì)譜鑒定,確認純化的蛋白確實是BmAPH基因的外源表達產(chǎn)物。(5)通過RT-PCR檢測BmAPH基因的表達模式,結果發(fā)現(xiàn)BmAPH基因在被檢測的9個組織和23個發(fā)育時間點均有表達,且?guī)缀鯖]有組織或時期表達特異性。以純化的蛋白作為抗原免疫小鼠,制備多克隆抗體。以提取的大造5齡3天幼蟲的總蛋白作為抗原,通過Western blotting驗證,結果表明制備的多克隆抗體是特異性的,可用于后續(xù)的實驗。對大造5齡3天幼蟲頭和中腸進行免疫組織化學分析,結果表明BmAPH存在于基膜中�；げ粌H對細胞、組織結構起支持作用,同時也是滲透性的障礙,可調(diào)節(jié)分子和細胞的運動,一定程度上可使細胞或組織免受損傷。(6)純化蛋白的酶活測定表明,BmAPH具有乙酰肽水解酶活性。通過測定不同反應溫度或同一溫度下不同pH對酶活性的影響,發(fā)現(xiàn)BmAPH活性易受溫度和pH的影響,最適反應溫度和最適pH值分別為50℃和7.7。分析三種有機磷殺蟲劑(毒死蜱、馬拉硫磷和辛硫磷)對BmAPH活性的影響,發(fā)現(xiàn)它們均能抑制BmAPH的活性。馬拉硫磷和辛硫磷對BmAPH活性的抑制中濃度IC50分別為7.62 mg/L和7.90 mg/L,相對而言,毒死蜱對BmAPH活性的抑制程度更高,IC50為1.60 mg/L。同時,測定用有機磷殺蟲劑處理家蠶后整蠶和中腸中APH的活性。與對照相比,結果表明分別用殺蟲劑處理家蠶后整蠶或中腸中APH的活性顯著地降低了。綜上所述,從家蠶基因組中鑒定出9個脯氨酰寡肽酶家族候選基因,與其他物種脯氨酰寡肽酶家族基因進行了比較分析,并從分子水平檢測了該家族基因在家蠶不同組織以及不同發(fā)育時期的表達模式,這為研究脯氨酰寡肽酶家族基因在家蠶及其他昆蟲中的潛在功能或進化關系提供了一定依據(jù)。另外,對BmAPH進行了克隆、表達模式、組織定位和酶活性分析,為研究其他昆蟲中乙酰肽水解酶的功能提供了有用信息。重要的是,本研究結果表明有機磷殺蟲劑能抑制BmAPH的活性,說明BmAPH是有機磷殺蟲劑的敏感靶標。
[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.
【學位授予單位】：重慶大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q55;Q78
，
本文編號：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.
【學位授予單位】：重慶大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q55;Q78
，
本文編號：2139860
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