家蚕丝氨酸蛋白酶BmHP14基因的克隆与表达模式分析

家蚕受到外源微生物侵染或损伤时,前酚氧化酶原级联反应中的起始丝氨酸蛋白酶会激活下游信号通路,最终产生黑色素。采用RACE技术,获得了家蚕前酚氧化酶原级联反应起始丝氨酸蛋白酶——血淋巴蛋白酶编码基因的全长cDNA序列,命名为BmHP14(GenBank登录号:JQ954757)。BmHP14 cDNA全长2 508 bp,开放阅读框为2 013 bp,编码670个氨基酸,预测蛋白质分子质量71 kD,等电点5.09,N端17个氨基酸预测为信号肽序列。多重序列比对显示BmHP14与烟草天蛾HP14的相似度很高,达到57%;分子进化树中二者也聚为一支。RT-PCR分析表明,BmHP14在家蚕5龄第3天幼虫脂肪体、马氏管、精巢、卵巢、表皮、血细胞、头部均有表达,其中以脂肪体中的表达水平最高。以黑胸败血芽孢杆菌、大肠杆菌、球孢白僵菌注射侵染家蚕5龄第3天幼虫,Real-time PCR检测显示在受到病菌侵染后,幼虫脂肪体中的BmHP14表达上调。Westernblotting检测结果显示,BmHP14在家蚕体液中以前体和成熟体的形式共同存在。研究结果提示,BmHP14是家蚕前酚氧化酶原级联反应信号通路中的关键酶。     

丝氨酸蛋白酶在昆虫先天免疫中的功能和作用机制研究进展

昆虫先天免疫系统包括细胞免疫和体液免疫。细胞免疫主要通过昆虫血液中的浆细胞对入侵的微生物进行吞噬;体液免疫比较复杂,涉及到昆虫体内许多蛋白质之间的相互作用,其中丝氨酸蛋白酶及其同源物与血淋巴黑化和抗菌肽合成等过程密切相关,如参与胞内酚氧化酶原及Toll免疫信号途径的激活等,因而在昆虫的体液免疫中发挥着重要作用。本文就昆虫丝氨酸蛋白酶及其同源物的类型、结构与功能以及家蚕丝氨酸蛋白酶的研究进展进行综述,为促进家蚕、蜜蜂等经济昆虫先天免疫的分子机制研究提供参考。     

家蚕丝氨酸蛋白酶抑制剂Bmserpin 6的原核表达及对酚氧化酶原活性和抗菌肽表达的调控作用

丝氨酸蛋白酶抑制剂(serpin)对昆虫的免疫反应起重要调节作用。根据家蚕基因组数据库序列设计引物,从家蚕幼虫血细胞中克隆了Bmserpin6的编码基因,并构建重组表达载体,在大肠杆菌中成功表达了重组Bmserpin6蛋白。将纯化的重组Bmserpin6注射家蚕5龄幼虫后6 h提取家蚕血液制备血清,检测血清样品中的酚氧化酶原活性显著下降(P0.05)。体腔注射重组Bmserpin6后的家蚕5龄幼虫再通过微球菌诱导蚕体内抗菌肽gloverin2基因的表达,结果显示幼虫脂肪体和血细胞中的gloverin2基因表达显著下调(P0.05)。依据上述结果推测:体外表达的重组Bmserpin6可以调节蚕体内2个重要的免疫途径,即抑制蚕体酚氧化酶原的激活和外源细菌诱导的蚕体抗菌肽的产生。     

家蚕各组织酚氧化酶原基因的表达谱分析

酚氧化酶在昆虫体液免疫反应中起着非常重要的作用。根据GenBank上登录的家蚕酚氧化酶原基因的cDNA序列设计特异引物,通过半定量BT-PCR技术检测了家蚕各组织酚氧化酶原基因的表达谱,发现该基因的转录表达具有组织特异性。我们的实验结果为进一步研究家蚕酚氧化酶原基因的功能提供了分子基础。     

参与家蚕免疫反应的clip丝氨酸蛋白酶家族基因分析

丝氨酸蛋白酶在昆虫的新陈代谢和生长发育等多种生理过程中起重要作用,特别是含clip结构域的丝氨酸蛋白酶与昆虫的免疫级联激活途径密切相关。为探索家蚕clip丝氨酸蛋白酶在家蚕先天免疫反应信号通路中的作用,对家蚕(Bombyxmori)与烟草天蛾(Manduca sexta)中鉴定获得的含有clip结构域的丝氨酸蛋白酶进行序列比对和系统进化分析,发现BmSP78与MsPAP-1、BmSP127与MsHP8、BmSP124与MsHP1、BmSP125与MsHP17位于同一进化支上,4对clip丝氨酸蛋白酶的氨基酸序列相似度分别为70%、68%、81%和57%,另外的8个蛋白酶BmSP23、BmSP111、BmSP95、BmSP135、BmSP129、BmSP137、BmSP91和BmSP99构成了家蚕特有的一群clip丝氨酸蛋白酶。用革兰阴性细菌沙雷氏菌和革兰阳性细菌黑胸败血菌分别注射感染家蚕5龄第4天幼虫后,通过半定量RT-PCR检测分析clip丝氨酸蛋白酶基因在不同感染时间点的表达特征:病原菌诱导后蚕体中有11个clip丝氨酸蛋白酶基因mRNA转录水平有明显变化,其中BmSP102和BmSP96于黑胸败血菌诱导12 h后在血细胞中明显上调表达,而BmSP125在沙雷氏菌和黑胸败血菌感染12 h后的脂肪体中均明显上调表达。研究结果为建立家蚕clip丝氨酸蛋白酶可能参与的免疫级联反应路径提供了重要线索。     

家蚕和野桑蚕酚氧化酶原基因的组织表达差异比较

酚氧化酶在昆虫体液免疫反应中起着非常重要的作用。根据GenBank中登录的家蚕和野桑蚕酚氧化酶原基因cDNA序列设计特异引物,用半定量RT-PCR方法检测了家蚕和野桑蚕各组织中酚氧化酶原基因(PPO1和PPO2)的表达谱,发现该基因在家蚕和野桑蚕组织中的表达差异较大,且具有明显的组织特异性。家蚕PPO1基因只在血液中被检出有很高的表达,而野桑蚕PPO1基因在血液和头部中均有表达,且在血液中的表达量最高;PPO2基因在家蚕组织中的表达量从高至低顺序为血液、脂肪体、卵巢、头部、表皮、精巢、中肠和丝腺,而在野桑蚕组织中的表达量从高至低顺序为血液、头部、中肠、表皮、精巢、卵巢和脂肪体,在丝腺中没有被检出有表达。推测可能是由于起源于野桑蚕的家蚕在长期的驯化及人工选择过程中引起了酚氧化酶原基因功能的变化。     

家蚕胚胎各发育时期酚氧化酶原基因的转录活性分析

酚氧化酶在昆虫体液免疫反应中起着非常重要的作用。利用GenBank上登录的家蚕酚氧化酶原基因的cDNA序列设计特异引物．通过半定量RT—PCR技术检测了家蚕胚胎各发育时期（从未受精卵到产卵后第9d）酚氧化酶原基因的转录活性,发现该基因的转录表达具有时空特异性。我们的试验结果为进一步研究家蚕酚氧化酶原基因的功能提供了新的线索。     

家蚕丝氨酸蛋白酶抑制剂基因serpin16的表达规律及体外重组表达

家蚕(Bombyxmori)的丝腺是丝蛋白合成分泌的场所,存在多种丝氨酸蛋白酶抑制剂。为探究家蚕丝蛋白的合成和保护机制,采用半定量RT-PCR的方法调查家蚕丝氨酸蛋白酶抑制剂基因serpin16在家蚕不同发育时期和5龄第5天幼虫各个组织器官中的表达特征,结果表明家蚕serpin16基因仅在4眠-5龄第6天的发育期表达,并且仅在丝腺中特异表达,其中在中部丝腺前区转录水平最高,而在中部丝腺中区和后区的转录水平较低;进一步构建pGEX-4T-1-serpin16原核表达载体,并转化至大肠杆菌(Eschevichia coli)BL21(DE3),经IPTG诱导获得融合蛋白,纯化后得到单一的目的蛋白。家蚕serpin16基因在幼虫丝腺的特异表达模式提示其可能与家蚕的吐丝过程密切相关,推测该基因在维持丝腺稳定的泌丝环境中发挥重要作用。     

酚氧化酶在家蚕血淋巴中的活力分布及影响因素的研究

昆虫血淋巴黑化的形成由激活酚氧化酶原的级联系统所引发,酚氧化酶在昆虫体液免疫中起着非常重要的作用。本试验以家蚕3龄末期开始每天抽取血淋巴制成粗酶液,以L-DOPA为底物,测定各龄期活力分布规律,并且通过改变酚氧化酶与底物反应的条件,发现温度与PH值对酚氧化酶添力具有很大的影响。     

柞蚕丝氨酸蛋白酶抑制剂6基因Apserpin-6的克隆及功能分析

昆虫的丝氨酸蛋白酶抑制剂(serpin)可以通过抑制丝氨酸蛋白酶的活性调控机体的免疫防御反应。依据柞蚕中肠转录组数据库中一段编码serpin的CDS序列设计正、反向引物,以柞蚕幼虫总c DNA为模板克隆得到一段全长1 239 bp的序列,经序列分析后命名为Apserpin-6(Gen Bank登录号:MF944108)。该基因编码412个氨基酸残基,其中N端第1~17位氨基酸为信号肽序列,在C端第357~391位氨基酸之间有一个反应中心环,第374~375位丝氨酸之间是预测的蛋白质裂解位点。半定量RT-PCR检测Apserpin-6在柞蚕5龄幼虫脂肪体中的表达量最高,在血淋巴和头部组织的表达量次之,在中肠、丝腺和马氏管中的表达量较低。荧光定量PCR检测在受到柞蚕肠球菌(Enterococcus pernyi)、白僵菌(Beauveria bassiana)以及柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedrovirus)侵染后的柞蚕幼虫血淋巴中,Apserpin-6的表达量均明显上升。利用在原核表达系统表达的重组Apserpin-6蛋白,对柞蚕幼虫血淋巴中的酚氧化酶原(pro PO)和酚氧化酶(PO)活性进行体外抑制试验,结果显示该重组蛋白能够抑制pro PO的活性,但对PO活性没有影响。试验结果提示,Apserpin-6可能作为一种负调控因子参与调控柞蚕血淋巴对于病原菌的免疫防御反应。     

A trypsin-like serine protease is involved in pseudorabies virus invasion through the basement membrane barrier of porcine nasal respiratory mucosa

ABSTRACT: Several alphaherpesviruses breach the basement membrane during mucosal invasion. In the present study, the role of proteases in this process was examined. The serine protease-specific inhibitor AEBSF inhibited penetration of the basement membrane by the porcine alphaherpesvirus pseudorabies virus (PRV) by 88.1% without affecting lateral spread. Inhibitors of aspartic-, cysteine-, and metalloproteases did not inhibit viral penetration of the basement membrane. Further analysis using the Soybean Type I-S trypsin inhibitor for the serine protease subcategory of trypsin-like serine proteases resulted in a 96.9% reduction in plaque depth underneath the basement membrane. These data reveal a role of a trypsin-like serine protease in PRV penetration of the basement membrane.     

Serine protease activity in excretory-secretory products of Oestrus ovis (Diptera: Oestridae) larvae

The sheep bot fly, Oestrus ovis, is a very common myiasis of nasal and sinus cavities of sheep and goats causing severe welfare and production implications. As the viability of O. ovis adult flies strictly depends on larval abilities to assimilate and to stock nutrients from the host, it was necessary to investigate proteolytic activities in larval excretory/secretory products (ESP). ESP of O. ovis larvae degrade mucosal and plasmatic components such as mucin, albumin or immunoglobulin G. A preliminary biochemical characterization, using substrate gel analysis and inhibitor sensitivity, demonstrated the presence of at least six major serine proteases (molecular weights from 20 to 100 kDa), mainly trypsin-like, secreted in the digestive tube of larvae. Their involvement in larval trophic activity and evasion from the host immune response is further discussed as O. ovis excretory/secretory serine proteases could represent potential vaccinal targets.     

Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization

Mammalian fertilization requires sperm to penetrate the cumulus to reach the oocyte. Although sperm hyaluronidase has long been believed to participate in the penetration process, our previous works revealed that neither of two sperm hyaluronidases, SPAM1 and HYAL5, are essential for fertilization. In this study, we have produced double-knockout mice lacking SPAM1 and either one of two sperm serine proteases, ACR and PRSS21, and characterized the mutant sperm. The SPAM1/ACR- and SPAM1/PRSS21-deficient males were fertile, whereas epididymal sperm of the mutant mice exhibited a reduced capacity to fertilize the oocytes in vitro. Despite normal motility, the ability of sperm to traverse the cumulus matrix was more severely impaired by the loss of SPAM1 and ACR or SPAM1 and PRSS21 than by the loss of only SPAM1. Moreover, SPAM1/ACR- and SPAM1/PRSS21-deficient sperm accumulated on the surface (outer edge) of the cumulus more abundantly than SPAM1-deficient sperm. These results suggest that ACR or PRSS21 or both may function cooperatively with SPAM1 in sperm/cumulus penetration.     

Involvement of proteases in porcine reproductive and respiratory syndrome virus uncoating upon internalization in primary macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in differentiated macrophages. In macrophages, heparan sulphate glycosaminoglycans mediate the initial PRRSV attachment and the receptor sialoadhesin mediates both PRRSV attachment and internalization into endosomes. Upon a pH drop, PRRSV is uncoated and its genome is released from the endosomes into the cytoplasm, which allows virus replication. However, expression of heparan sulphate and sialoadhesin in non-susceptible cells only allows virus internalization, but no virus uncoating and infection, indicating that other factors are involved. In the present study, it is shown that treatment of macrophages with serum (mainly the alpha-globulin fraction) inhibited PRRSV infection without affecting attachment and internalization. Because alpha-globulins contain several protease inhibitors, macrophages were treated with different protease inhibitors to investigate the involvement of proteases in PRRSV uncoating. Treatment of macrophages with broadly active inhibitors of serine or aspartic proteases, but not cysteine- or metallo-proteases, inhibited PRRSV uncoating and infection. Further investigation using specific inhibitors indicated that the aspartic protease cathepsin E is involved during PRRSV uncoating, but did not allow identification of the serine protease involved. The involvement of cathepsin E during PRRSV uncoating was confirmed by partial co-localization of internalized PRRSV with cathepsin E. Furthermore, cathepsin E expression increased with macrophage cultivation, which was positively correlated with an increased susceptibility to PRRSV infection. Together, these data show that, in macrophages, both the aspartic protease cathepsin E and an unidentified trypsin-like serine protease are involved in uncoating of internalized PRRSV and subsequent infection.     

Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken.

A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.     

Temporal relationships and characterisation of extracellular proteases from benign and virulent strains of Bacteroides nodosus as detected in zymogram gels

Extracellular proteases produced by Bacteroides nodosus in a peptone rich modified trypticase-arginine-serine broth medium were separated and characterised by relative mobility (Rf) in electrophoretic zymogram gels. One benign and two virulent protease banding patterns were established with isolates from sheep, cattle and goats. They correlated with other laboratory tests for virulence but were independent of serogroup. The electrophoretic zymogram method was unable to differentiate intermediate from virulent strains. The time required for the production of maximum levels and numbers of protease bands was four to five days for benign and five to six days for virulent B nodosus. Elevated temperatures (above 45 degrees C) and pH extremes (below pH 6 and above pH 9) modified the electrophoretic banding patterns. The molecular weights of the proteases ranged from 8000 to 43,000 daltons and the isoelectric points from pH 4.90 to 5.90. They are serine proteases and this property can be utilised in affinity purification of these molecules.     

家蚕Kazal型丝氨酸蛋白酶抑制剂基因BmSPI3的克隆与表达特征分析

Kazal型丝氨酸蛋白酶抑制剂(SPI)在机体的发育、免疫等生理过程中发挥重要的作用。从家蚕蛹cDNA文库中获得一条全长为728bp的基因序列,对该基因编码的氨基酸序列进行同源性比对,发现其蛋白具有保守的Kazal型丝氨酸蛋白酶抑制剂结构域(CⅠ-X3-CⅡ-X8-CⅢ-X14-CⅣ-X6-CⅤ-X13-CⅥ),且P1活性位点为赖氨酸(K),因此将该基因命名为BmSPI3(Gen-Bank登录号:DN237641)。通过PCR扩增BmSPI3基因,经BamHⅠ和XhoⅠ双酶切后成功构建了重组表达质粒pGEX-4T-1-BmSPI3,并转化到E.coliBL21(DE3)表达,经SDS-PAGE电泳检测在约36kD处有明显的蛋白表达条带。提取各发育时期蚕体和5龄幼虫各个组织的总RNA进行荧光定量PCR,检测结果表明:BmSPI3在5龄幼虫期表达量最高,卵期最低;在5龄幼虫表皮中的表达量最高,在马氏管的表达量最低。推测BmSPI3对胰蛋白酶和类胰蛋白酶具有抑制作用。