家蚕丝氨酸蛋白酶抑制剂（Bmserpin-2）和类成虫盘生长因子（BmIDGF）的亚细胞定位及组织表达分析
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摘要
家蚕是重要的经济昆虫,蚕丝业是我国拥有5000多年历史的传统产业,在社会经济文化生活中占有重要地位,同时家蚕也是鳞翅目的模式昆虫。家蚕功能基因组学的研究将为全面、准确的了解家蚕生物性状的遗传基础和分子调控机制,为改善蚕丝质量提供理论基础,也为揭示基础生物学现象奠定了基础。
本论文课题通过实验和生物信息学相结合的方法克隆得到了家蚕二个基因,并对这些基因做了初步的结构和功能分析。主要研究结论如下:
(1)从Genbank中搜索到家蚕Bmserpin-2基因(GenBank登录号:AF242200),并运用相关的生物信息学软件对其进行分析,表明家蚕Bmserpin-2基因编码374个氨基酸,具有保守的SERPIN结构域,在进化上与其它无脊椎动物的serpin-2具有高度同源性。RT-PCR结果显示该基因在家蚕卵巢、精巢、脂肪体、丝腺、中肠、马氏管、血淋巴,以及蚕卵、一龄到五龄的家蚕中都有表达。利用荧光定量PCR分析在感染家蚕核型多角体病毒(BmNPV)后不同时间段该基因在中肠表达情况,结果表明:抗性家蚕品系(NB)在感染BmNPV后,各时间段Bmserpin-2的表达量无显著性差异。感性家蚕品系(306)在感染BmNPV 72h后,Bmserpin-2的表达量显著提高,表明Bmserpin-2可能参与了家蚕免疫。
利用pET-30a表达系统,在大肠杆菌中表达该基因,运用质谱技术对其进行鉴定,并纯化蛋白制备抗体。通过细胞免疫组化实验发现该基因定位于整个细胞中。
(2)从Genbank中搜索到家蚕IDGF基因(GenBank登录号:AB183872),即BmIDGF,并运用相关的生物信息学软件对其进行了分析,表明BmIDGF基因编码434个氨基酸,具有保守的glycosylhydrolases family 18结构域。BmIDGF属于几丁质酶18家族中的GroupⅤ,在进化上与其它无脊椎动物的IDGF蛋白具有高度同源性。RT-PCR结果显示该基因在家蚕卵巢、精巢、脂肪体、丝腺、中肠、马氏管、血淋巴中,家蚕蚕卵的各个发育时期,以及一龄到五龄的家蚕中都有表达,Western blot的结果也进一步验证了该基因在家蚕血淋巴、中肠、卵巢、丝腺和脂肪体中的表达。利用pET-30a表达系统,在大肠杆菌中表达了该基因,运用质谱技术对其进行了鉴定,并纯化蛋白制备了抗体。通过细胞免疫组化实验发现该基因编码的蛋白可能为分泌型蛋白。
Silkworm (Bombyx mori) is an important economic insect and is regarded as a model insect of Lepidoptera. Silkworm genome was sequenced by the whole-genome shotgun method, and released to GenBank in the October of 2003. This is a significant development of silkworm research, and lay the foundation for the study of functional genes of silkworm.
Using the method comprised experiment and bioinformatics approaches, we cloned two novel genes of B. mori, and made some basical analysis of these genes in the research. The research process and the main conclusions are present as follows:
(1) Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding B. mori serpin-2 {Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.
(2) In this study, we cloned a novel IDGF gene in B. mori and designated it as BmIDGF. We found that the BmIDGF gene contains eight exons and seven introns, encoding a peptide of 434 amino-acid residues. The protein was predicted to contain one conserved motif of the glycosyl hydrolases family 18 and fall into groupⅤchitinases. Sequence alignment showed that BmIDGF shares extensive homology with other invertebrate IDGF. RT-PCR analysis showed that BmIDGF is expressed in all developmental stages of silkworm larvae and various larvae tissues, which was further confirmed by Western blot analysis. Subcellular localization analysis indicated that BmIDGF is located in the extracellular space. We also successfully expressed it in E. coli and further characterized it by SDS-PAGE and mass spectrometry. Taken together, our data suggests that BmIDGF is a chitinase- like extracellular protein, and provides an excellent platform for subsequent studies on its enzyme activity and role in B. mori development.
本论文课题通过实验和生物信息学相结合的方法克隆得到了家蚕二个基因,并对这些基因做了初步的结构和功能分析。主要研究结论如下:
(1)从Genbank中搜索到家蚕Bmserpin-2基因(GenBank登录号:AF242200),并运用相关的生物信息学软件对其进行分析,表明家蚕Bmserpin-2基因编码374个氨基酸,具有保守的SERPIN结构域,在进化上与其它无脊椎动物的serpin-2具有高度同源性。RT-PCR结果显示该基因在家蚕卵巢、精巢、脂肪体、丝腺、中肠、马氏管、血淋巴,以及蚕卵、一龄到五龄的家蚕中都有表达。利用荧光定量PCR分析在感染家蚕核型多角体病毒(BmNPV)后不同时间段该基因在中肠表达情况,结果表明:抗性家蚕品系(NB)在感染BmNPV后,各时间段Bmserpin-2的表达量无显著性差异。感性家蚕品系(306)在感染BmNPV 72h后,Bmserpin-2的表达量显著提高,表明Bmserpin-2可能参与了家蚕免疫。
利用pET-30a表达系统,在大肠杆菌中表达该基因,运用质谱技术对其进行鉴定,并纯化蛋白制备抗体。通过细胞免疫组化实验发现该基因定位于整个细胞中。
(2)从Genbank中搜索到家蚕IDGF基因(GenBank登录号:AB183872),即BmIDGF,并运用相关的生物信息学软件对其进行了分析,表明BmIDGF基因编码434个氨基酸,具有保守的glycosylhydrolases family 18结构域。BmIDGF属于几丁质酶18家族中的GroupⅤ,在进化上与其它无脊椎动物的IDGF蛋白具有高度同源性。RT-PCR结果显示该基因在家蚕卵巢、精巢、脂肪体、丝腺、中肠、马氏管、血淋巴中,家蚕蚕卵的各个发育时期,以及一龄到五龄的家蚕中都有表达,Western blot的结果也进一步验证了该基因在家蚕血淋巴、中肠、卵巢、丝腺和脂肪体中的表达。利用pET-30a表达系统,在大肠杆菌中表达了该基因,运用质谱技术对其进行了鉴定,并纯化蛋白制备了抗体。通过细胞免疫组化实验发现该基因编码的蛋白可能为分泌型蛋白。
Silkworm (Bombyx mori) is an important economic insect and is regarded as a model insect of Lepidoptera. Silkworm genome was sequenced by the whole-genome shotgun method, and released to GenBank in the October of 2003. This is a significant development of silkworm research, and lay the foundation for the study of functional genes of silkworm.
Using the method comprised experiment and bioinformatics approaches, we cloned two novel genes of B. mori, and made some basical analysis of these genes in the research. The research process and the main conclusions are present as follows:
(1) Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding B. mori serpin-2 {Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.
(2) In this study, we cloned a novel IDGF gene in B. mori and designated it as BmIDGF. We found that the BmIDGF gene contains eight exons and seven introns, encoding a peptide of 434 amino-acid residues. The protein was predicted to contain one conserved motif of the glycosyl hydrolases family 18 and fall into groupⅤchitinases. Sequence alignment showed that BmIDGF shares extensive homology with other invertebrate IDGF. RT-PCR analysis showed that BmIDGF is expressed in all developmental stages of silkworm larvae and various larvae tissues, which was further confirmed by Western blot analysis. Subcellular localization analysis indicated that BmIDGF is located in the extracellular space. We also successfully expressed it in E. coli and further characterized it by SDS-PAGE and mass spectrometry. Taken together, our data suggests that BmIDGF is a chitinase- like extracellular protein, and provides an excellent platform for subsequent studies on its enzyme activity and role in B. mori development.
引文
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[2]Hunt LT,Dayhoff MO.A surprising new protein superfamily containing ovalbumin,antithrombin-Ⅲ,and alpha 1-proteinase inhibitor.Biochemical and biophysical research communications.1980(95):864-71.
[3]Laskowski MJ,Kato I.Protein inhibitors of proteinases.Annu Rev Biochem.1980(49):593-626.
[4]Milstone AM,Harrison LM,et al.A broad spectrum Kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum.The Journal of biological chemistry.2000(275):29391-9.
[5]Shimomura T,Denda K,et al.Hepatocyte growth factor activator inhibitor,a novel Kunitz-type serine protease inhibitor.The Journal of biological chemistry.1997(272):6370-6.
[6]郑青亮,盛清,张耀洲.Kazal型蛋白酶抑制剂结构与功能研究进展.生物工程学报.2006(22):695-700.
[7]Friedrich T,Kroger B,et al.A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus.The Journal of biological chemistry.1993(268):16216-22.
[8]Arnd B.E.Brauer,Gonzalo J.Domingo,et al.A Conserved cis Peptide Bond Is Necessary for the Activity of Bowman-Birk Inhibitor Protein.Biochemistry.2002(41):10608-15.
[9]QI R-F,SONG Z-W,et al.Structural Features and Molecular Evolution of Bowman-Birk Protease Inhibitors and Their Potential Application.2005(37):283-92.
[10]Yavelow J FT,Kennedy AR.Bowman-Birk soybean protease inhibitor as an anticarcinogen.Cancer Res.1983(43):2454-9.
[11]Irving JA,Pike RN,et al.Phylogeny of the serpin superfamily:implications of patterns of amino acid conservation for structure and function.Genome research.2000(10):1845-64.
[12]Salzet M,Vieau D,et al.Serpins:an evolutionarily conserved survival strategy:Immunology today.1999(20):541-4.
[13]Huntington JA,Read RJ,et al.Structure of a serpin-protease complex shows inhibition by deformation.Nature.2000(407):923-6.
[14]Dementiev A,Dobo J,et al.Active site distortion is sufficient for proteinase inhibition by serpins:structure of the covalent complex of α1-proteinase inhibitor with porcine pancreatic elastase.The Journal of biological chemistry.2006(281):3452-7.
[15]Diana van Gent,Paul Sharp,et al.Serpins:structure,function and molecular evolution.The International Journal of Biochemistry and Cell Biology.2003(35):1536-47.
[16]Silverman GA,Bird PI,et al.The serpins are an expanding superfamily of structurally similar but functionally diverse proteins.Evolution,mechanism of inhibition,novel functions,and a revised nomenclature.The Journal of biological chemistry.2001(276):33293-6.
[17]Mans BJ,Louw AI,et al.Amino acid sequence and structure modeling of savignin,a thrombin inhibitor form the tick,Ornithodoros savignyi.Insect Biochem Mol Biol.2002(32):821-8.
[18]松杨.昆虫黑化反应的分子机制研究进展.国外医学寄生虫病分册.2003(30):164-8.
[19]Jiang H,Ma C,et al.Beta-1,3-glucan recognition protein-2(betaGRP-2)from Manduca sexta;an acute-phase protein that binds beta-1,3-glucan and lipoteichoic acid to aggregate fungi and bacteria and stimulate prophenoloxidase activation.Insect Biochem Mol Biol.2004(34):89-100.
[20]Fabrick JA,Baker JE,et al.Innate immunity in a pyralid moth:functional evaluation of domains from a beta-1,3-glucan recognition protein.The Journal of biological chemistry.2004(279):26605-11.
[21]时超美.昆虫酚氧化酶原活化及其在免疫中的作用.昆虫知识.2000(37):310-4.
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