Tissue Distribution and Purification of Prophenoloxidase in Larvae of Asian Corn Borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

Phenoloxidase (PO) plays an important role in the de-fense response to the foreign invaders, e.g. pathogens andparasites, in the formation of melanin as well as in cuticlesclerotization in insects [1]. PO is synthesized as azymogen, prophenoloxidase (PPO)…     

亚洲玉米螟幼虫血清中酚氧化酶原的性

采用40%硫酸铵沉淀、Blue Sepharose CL-6B亲和层析和Phenyl Sepharose CL-4B疏水层析等方法,从亚洲玉米螟Ostrinia furnacalis (Guenée) 幼虫血清中分离纯化了酚氧化酶原。酚氧化酶原全酶相对分子量约为158 kD,亚基相对分子量约为80 kD和78 kD。酚氧化酶原为糖蛋白,该酶原易被0.1 mmol/L CPC (氯代十六烷基吡啶)、 50%甲醇、 1 mg/mL昆布多糖和1 mg/mL胰蛋白酶激活。该酶反应的最适pH为7.0,最适温度为25～30℃,Ca²⁺和Mg²⁺可增强该酶的活性。     

Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae

Prophenoloxidase (PPO) was isolated from the hemolymph of Ostrinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity were achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The N-terminal amino acid sequences of two subunits are as follows: PPO1, FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2, FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The amino acid composition of purified PPO was similar to that from Galleria mellonella. The enzyme kinetic property of the purified protein showed that the affinity of the enzyme for dopamine was higher than that for l-DOPA and N-acetyldopamine. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (beta-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA). Both Mg(2+) and Cu(2+) stimulated PO activity when compared with controls. The beta-sheet content of PPO treated with Mg(2+) and Cu(2+) increased significantly (P<0.05). The purified PPO has magnesium level of 5.674+/-2.294 microg/mg and copper level of 1.257+/-0.921 microg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.     

家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase, PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。     

黄粉甲幼虫抗菌物质的诱导及其抗菌活性

采用饥饿法、紫外线照射法和针刺法处理黄粉甲Tenebriomolitor 6龄幼虫后均能诱导其 产生抗菌物质,收集的血淋巴上清液对真菌有抑制作用,对细菌无抑制作用;经热处理后的血 淋巴上清液则对细菌有抑制作用,而对真菌无抑制作用。SDS-PAGE检测结果发现,与未诱导的 对照相比经诱导的黄粉甲幼虫血淋巴中,原有的一类大分子蛋白质如分子量分别为97kD、44 kD和37 kD左右的蛋白质缺失;而ESI-MS分析结果显示诱导后比诱导前黄粉甲幼虫血淋巴中有 小分子物质产生,推测可能是此类缺失蛋白质分解为小分子量的抗菌肽,从而表现出抗菌活性 。     

越冬松针瘿蚊幼虫整体携脂蛋白 的分离和纯化

采用KBr密度梯度超速离心并结合常规Sepharose CL-4B凝胶柱层析,从越冬松针瘿蚊Thecodiplosis japonensis(Uchida et Inouye) 幼虫整体中,分离并纯化了一种携脂蛋白。这是第二例从昆虫整体分离并纯化出携脂蛋白的报道。采用凝胶柱层析确定该携脂蛋白的相对分子质量为638 kD,它是由分别为240 kD和52 kD的两个亚基组成 。整体分子中含有52.8%的蛋白和47.2%的脂类 。苏丹黑B和希夫氏试剂染色显示阳性,说明它是一种糖脂复合蛋白。采用超速离心确定它的密度为1.11 g/mL,表明它是一种高密度的脂蛋白。     

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.     

Isolation and characterization of a group of isolectins with galactose/N-acetylgalactosamine specificity from hemolymph of the giant silk moth Hyalophora cecropia

A series of isolectins from Hyalophora cecropia was purified by affinity chromatography on d-GalNAc-Sepharose. The yield of the lectin was 0.4 mg/ml of hemolymph and the concentration was about the same in larvae and pupae. The molecular weight of the lectin was around 160,000 and the sizes of the subunits were 41 and 38 kD for the A and the B chains, respectively. The isolectins are believed to be tetramers with varying proportions between the two subunits. Inhibition studies indicate that the A chain has a binding site with specificity for d-Gal/d-GalNAc while the B chain has specificity for an unknown structure on rabbit erythrocytes not necessarily of carbohydrate nature. A d-Gal/d-GalNAc specific lectin was found also in Anthereae pernyi but the yield was only 0.03 mg/ml of hemolymph.     

Phenoloxidase in larvae of Plodia interpunctella (Lepidoptera: Pyralidae): molecular cloning of the proenzyme cDNA and enzyme activity in larvae paralyzed and parasitized by Habrobracon hebetor (Hymenoptera: Braconidae)

Phenoloxidase (PO) is a major component of the insect immune system. The enzyme is involved in encapsulation and melanization processes as well as wound healing and cuticle sclerotization. PO is present as an inactive proenzyme, prophenoloxidase (PPO), which is activated via a protease cascade. In this study, we have cloned a full-length PPO1 cDNA and a partial PPO2 cDNA from the Indianmeal moth, Plodia interpunctella (Hubner) (Lepidoptera: Pyralidae) and documented changes in PO activity in larvae paralyzed and parasitized by the ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae). The cDNA for PPO1 is 2,748 bp and encodes a protein of 681 amino acids with a calculated molecular weight of 78,328 and pI of 6.41 containing a conserved proteolytic cleavage site found in other PPOs. P. interpunctella PPO1 ranges from 71-78% identical to other known lepidopteran PPO-1 sequences. Percent identity decreases as comparisons are made to PPO-1 of more divergent species in the orders Diptera (Aa-48; As-49; and Sb-60%) and Coleoptera (Tm-58; Hd-50%). Paralyzation of host larvae of P. interpunctella by the idiobiont H. hebetor results in an increase in phenoloxidase activity in host hemolymph, a process that may protect the host from microbial infection during self-provisioning by this wasp. Subsequent parasitization by H. hebetor larvae causes a decrease in hemolymph PO activity, which suggests that the larval parasitoid may be secreting an immunosuppressant into the host larva during feeding.     

纤维素酶中具有壳聚糖水解酶活性成分的鉴定

在壳聚糖酶的研究过程中,目前已发现37种酶具有非专一性地降解壳聚糖的能力[1].对这些非专一性酶水解壳聚糖的机理有两种看法:一些人认为,由于这些酶大都来自商业酶制剂,未经过进一步的纯化,故有人认为其中所含的少量杂质可能是产生水解活力的原因;但也有人认为,在所有的酶制剂中都存在同一种杂质似乎是不可能的,因为这些酶来源于广泛的微生物、真菌、哺乳动物和植物等.众所周知,酶具有高度的专一性,即对所催化的反应和底物有严格的选择性,一种酶往往只能催化一种或一类反应;有如此多的不同种类的酶能非专一性地水解壳聚糖.因而探讨具有水解…     

家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

【目的】克隆家蚕Bombyx mori Wnt信号通路下游关键基因Pangolin isoforms A/H/I/S转录剪接体X3 (Pangolin X3),分析其序列和表达特征。【方法】从NCBI数据库检索家蚕Pangolin X3,根据其编码序列(coding sequence, CDS)设计引物,利用PCR从家蚕幼虫中肠和血淋巴中进行克隆并测序验证。利用SilkDB 3.0, SMART,多序列比对和系统发育树分析Pangolin X3的序列特征。利用qRT-PCR分析Pangolin X3在家蚕5龄第3天幼虫不同组织(头、血淋巴、体壁、性腺、中肠、前部丝腺、中部丝腺、后部丝腺、脂肪体和马氏管)中的相对表达水平。【结果】从家蚕幼虫中肠和血淋巴克隆了Pangolin X3(GenBank登录号：XM＿038020921)的CDS,其开放阅读框长1 560 bp,编码519个氨基酸残基,预测分子量为55.86 kD,预测等电点为7.53。Pangolin X3蛋白含有保守的β-catenin结合位点和HMG结构域,其氨基酸序列在不同的昆虫中比较保守,特别是与DNA结合的HMG结构域...     

Enhanced production of secretory beta1,3-N-acetylglucosaminyltransferase 2 fusion protein into hemolymph of Bombyx mori larvae using recombinant BmNPV bacmid integrated signal sequence

The enhanced secretion of beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) fusion protein into the hemolymph of Bombyx mori larvae was studied using a recombinant B. mori nucleopolyhedrovirus (BmNPV) bacmid integrating seven signal sequences. When the BmNPV bacmid encoding the signal sequences from the silkworm B. mori bombyxin (bx) and B. mori prophenoloxidase-activating enzyme (ppae) was injected into silkworm larvae, 56.1 and 51.5mU/ml beta3GnT, respectively, were secreted into the hemolymph of silkworm larvae. For bx, 97.3% of the total beta3GnT activity was secreted into hemolymph, and only 1.1% remained in the intestines of silkworm larvae. For ppae, 90.8% of the total beta3GnT activity was secreted to the hemolymph, but 7.8% remained in the intestines of silkworm larvae. Using the BmNPV bacmid encoding bx, the amount of secreted beta3GnT was 91mug per larva, which was 2.5% of the total amount of protein in the hemolymph.     

家蝇幼虫血淋巴中抗真菌肽的诱导方法比较及抗真菌活性

以未诱导组作为空白对照研究比较真菌诱导、超声诱导和热诱导家蝇Musca domestica 幼虫血淋巴初提液的抗真菌肽效果,比较各种诱导方法诱导后的幼虫存活率;用凝胶层析法和高效液相分离纯化热诱导家蝇3龄幼虫抗真菌肽,检测其抗白假丝酵母菌Candida albicans和新生隐球菌Cryptococcus neoformans活性;SDS-PAGE分析抗真菌肽的蛋白分子量范围。结果表明：3种诱导方法诱导后家蝇幼虫均产生具有明显抗真菌作用的抗真菌肽,其初提液抑菌圈大小没有明显差别;真菌诱导组和热诱导组幼虫存活率低于对照组,而超声诱导组与对照组相比则无明显差别。经分离纯化后,抗真菌肽仍具有较好的抗真菌活性;SDS-PAGE分析表明该抗真菌肽有效成分的蛋白分子量在14.4 kD以下。结果提示热诱导家蝇幼虫产生抗真菌肽是一种方便、有效的诱导方式。     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

颈双缘姬蜂毒液对寄主小菜蛾的免疫抑制作用

对颈双缘姬蜂Diadromus collaris (Gravenhorst)及其毒液引起寄主小菜蛾Plutella xylostella的一些生理效应进行了研究。结果表明,颈双缘姬蜂寄生寄主后可引起寄主小菜蛾蛹总血细胞及浆血细胞和颗粒血细胞数量的上升。寄生后1天观察,血细胞延展行为受到影响,表现在颗粒血细胞放射状丝的产生及浆血细胞伪足的形成受到抑制。通过毒液对寄主离体幼虫血细胞延展行为、形态及活性影响的研究,发现毒液抑制了寄主离体浆血细胞的延展,但对颗粒血细胞的影响不明显;毒液引起寄主浆血细胞和颗粒血细胞的破裂和死亡,毒液对寄主幼虫血淋巴酚氧化酶活性有一定的抑制作用,当反应至40、60及80 min时,毒液处理和未经毒液处理的寄主血淋巴在490 nm处的吸光值差异比较明显。对毒液蛋白成分的聚丙烯酰胺凝胶电泳分析发现,毒液中有9种多肽,分子量介于9～50.2 kD,其中50.2、30.5、28.2、25.1 和12.6 kD的多肽含量较高, 与其他蜂毒液的一些作用已知的蛋白条带相似,因而推测它们同样具有免疫及发育抑制作用。结果证明颈双缘姬蜂毒液能破坏寄主细胞及体液因子调节的免疫反应。     

沙漠生物结皮芽孢杆菌产胞外多糖的纯化及其絮凝性的研究

【目的】以新疆古尔班通古特沙漠的生物结皮为样品,通过培养、筛选、分离得到一株高产胞外多糖(EPS)的菌株XJ-27,对XJ-27菌株所产的胞外多糖进行分离纯化,并对其絮凝性进行研究。【方法】利用DEAE sepharose CL-6B阴离子层析和Sephadex G100凝胶层析的方法对胞外多糖进行纯化,通过紫外分析方法和高效凝胶渗透色谱进行纯度的测定,利用高效凝胶渗透色谱法(HP-GPC)测定其分子量,以高岭土为体系对其絮凝性进行研究。【结果】利用层析分离的方法共得到2个胞外多糖的组分,对其中一个组分进一步纯化,得到组分EPS-I。结果表明,EPS-I纯度较高,分子量为575 kD。同时对胞外多糖的絮凝性进行了研究,结果表明该胞外多糖对高岭土为体系的絮凝率为80.4%。【结论】菌株XJ-27产胞外多糖,其胞外多糖具有絮凝性,对该胞外多糖进行分离纯化后,得到分子量为575 kD的多糖组分EPS-I。     

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate.     

广西眼镜蛇毒磷酸二酯酶分离纯化及部分性质

磷酸二酯酶(PDE)广泛存在于各种蛇毒中,在响尾蛇科、蝰蛇科、眼镜蛇科和海蛇科的部分蛇毒中都存在着该酶.不同的蛇毒其PDE活力及含量有很大差异.即便在生物学分类上十分接近的蛇种,其PDE活力亦相差甚远.该酶是核酸结构分析的一种重要工具酶,具有核酸水解酶作用,是一种核酸外切酶,它能从3'-端顺序地降解核糖或脱氧核糖多核苷酸,生成5'-单核苷酸.该酶既能水解DNA又能水解RNA,因而在核酸的鉴定和序列测定方面获得了广泛的应用.由于蛇毒PDE在核酸顺序测定中显示的重要性,因而对各种蛇毒纯化PDE的研究工作引起了国内外学者的广泛兴趣.王殿洪等[1]曾对江西蝮蛇毒中PDE的分离纯化及某些性质做过报道,但广西眼镜蛇毒中PDE的分离纯化及性质在国内外均未有报道.广西有丰富的眼镜蛇毒资源,而且有高活性的PDE.蛇毒PDE的生物学活性,国外研究得较少.它没有出血、凝血、纤溶及致死作用.蛇毒PDE水解核糖核酸产生5'-单核苷酸,5'-单核苷酸在医药上均有十分重要作用,制成药物治疗白血病和血小板减少症等.     

造血器官机能障碍后家蚕血淋巴中蛋白质成分的变化

为了研究家蚕Bombyx mori造血器官机能障碍后其血淋巴中蛋白质成分的变化,利用重离子射线局部照射家蚕幼虫的造血器官,检测了照射后家蚕血淋巴中的蛋白质成分及注射大肠杆菌后在体内诱导出现的应急蛋白量的变化。结果表明,照射蚕血淋巴中的蛋白质含量与对照蚕之间没有明显的差异。但在成分分析时发现,5龄起蚕血淋巴中70 kD附近的3条蛋白质谱带比对照蚕的浓度要高,随着个体的发育两者的浓度都上升;5龄后期则相反,对照蚕的浓度比照射蚕高;脂肪体中贮藏蛋白质的含量具有相似的变化趋势。用家蚕贮藏蛋白质SP-1及SP-2的抗血清进行免疫印迹反应的结果显示：70 kD附近的3条蛋白质谱带的最上面的一条为贮藏蛋白质SP-1,下面的二条为贮藏蛋白质SP-2;同时照射蚕血淋巴中分子量约为24 kD的蛋白质成分也发生变化,5龄前期的浓度比对照蚕低,5龄第3天几乎检测不到;全体照射与造血器官局部照射蚕之间的结果相似。照射蚕注射大肠杆菌后在体内诱导出现的应急蛋白量明显比对照蚕要少。由此认为家蚕幼虫造血器官与血淋巴中的蛋白质成分有关,造血器官的机能障碍、血球的数量减少可影响脂肪体中蛋白质的合成,从而使存在于血淋巴中的蛋白质成分发生变化。     

Iron binding proteins and their roles in the tobacco hornworm,Manduca sexta (L.)

Summary Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When⁵⁹Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle.Studies of the hemolymph disclosed two ironcontaining proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 g of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin inManduca sexta, the latter serving a storage function during development and possibly also a transport function.