蚯蚓纤溶酶分子生物学进展

蚯蚓纤溶酶是近年发现的一种新型的溶解血栓物质,属丝氨酸蛋白酶,不同种属的蚯蚓中均可分离到,具纤溶活性和溶栓活性。有较好的热稳定性,多为单体酶,多数兼有纤溶活性和纤溶酶原激活活性。不同种属的蚯蚓分离的纤溶酶性质上有一定差别。已获得多种纤溶酶的N端序列及部分核酸序列,相互之间及与某些蛋白酶之间有一定的同源性。纤溶酶通过降解目的蛋白的特定位点而起作用 。     

蚯蚓纤溶酶分子生物学进展

蚯蚓纤溶酶是近年发现的一种新型的溶解血栓物质,属丝氨酸蛋白酶,不同种属的蚯蚓中均可分离到,具纤溶活性和溶栓活性。有较好的热稳定性,多为单体酶,多数兼有纤溶活性和纤溶酶原激活活性。不同种属的蚯蚓分离的纤溶酶性质上有一定差别。已获得多种纤溶酶的N端序列及部分核酸序列,相互之间及与某些蛋白酶之间有一定的同源性。纤溶酶通过降解目的蛋白的特定位点而起作用。     

蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证

利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓(Lumbricusrubellus)中分离的F-Ⅰ-0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae的dbEST中获得该组分的核酸序列信息,设计特异引物,经过RT-PCR,成功地从赤子爱胜蚓(Eiseniafoetida)中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp,编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40%,因此为蚯蚓纤溶酶中的一个新基因,GenBank登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。     

重组海蚯蚓纤溶酶的克隆、表达及活性鉴定北大核心CSCD

纤溶酶在溶栓治疗中起重要作用,能够溶解血凝块的主要成分纤维蛋白。采用RACE方法从海蚯蚓消化道组织中扩增出海蚯蚓纤溶酶编码序列,构建该基因原核表达载体,并进一步构建工程菌表达融合蛋白,经Ni2+树脂柱纯化后通过平板法检测该融合蛋白纤维蛋白酶原激活活性。结果获得海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并成功构建重组表达载体p ET-21a-AFE,表达纯化出融合蛋白,该融合蛋白能够激活纤维蛋白酶原而溶解纤维蛋白。总之,本研究获得了海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并初步证明其具有纤维蛋白酶原激活活性,为临床新型溶栓药物的开发提供实验基础。     

重组海蚯蚓纤溶酶的克隆、表达及活性鉴定

纤溶酶在溶栓治疗中起重要作用,能够溶解血凝块的主要成分纤维蛋白。采用RACE方法从海蚯蚓消化道组织中扩增出海蚯蚓纤溶酶编码序列,构建该基因原核表达载体,并进一步构建工程菌表达融合蛋白,经Ni2+树脂柱纯化后通过平板法检测该融合蛋白纤维蛋白酶原激活活性。结果获得海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并成功构建重组表达载体p ET-21a-AFE,表达纯化出融合蛋白,该融合蛋白能够激活纤维蛋白酶原而溶解纤维蛋白。总之,本研究获得了海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并初步证明其具有纤维蛋白酶原激活活性,为临床新型溶栓药物的开发提供实验基础。     

Eisenia fetida中蚯蚓纤溶酶P-Ⅲ组分序列多态性

为了探讨蚯蚓纤溶酶P—Ⅲ组分序列多态性及其与蚓种关系。从Eisenia fetida中提取总RNA,利用P-Ⅲ组分序列两端的保守性设计引物,通过RT—PCR获得P—Ⅲ组分基因,并构建该组分质粒（pUC18）文库,经PCR筛选后进行序列测定及分析。在原已获得的Efp-Ⅲ-1和Efp-Ⅲ-3的基础上,利用中间引物从文库中筛选到Efp-Ⅲ-2基因。序列分析表明,该基因编码序列为720bp,编码239个氨基酸,与GenBank报道的点Efp-Ⅲ-2五条序列整体相似性高达99．02％。通过序列比对,发现Efb-Ⅲ-1、2和3具有完全相同的结构域,差异位点零星分布在序列中间;Lumbricus rubellus的六条序列同样具有Eisenia fetida这个特点,并无明显差异。序列比对证明它们是蚯蚓体内P-Ⅲ组分基因多态性的表现,而且在Lumbricidae科中与蚓种无关。     

蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证

利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓（Lumbricus rubellus）中分离的FⅠ0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae 的dbEST中获得该组分的核酸序列信息,设计特异引物, 经过RT-PCR,成功地从赤子爱胜蚓（Eisenia foetida）中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp, 编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40％,因此为蚯蚓纤溶酶中的一个新基因,GenBank 登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。     

蛇毒纤溶酶的神经生长因子活性

目的证明蛇毒生长因子具有神经生长因子活性,并初步探讨其原因.方法分实验组和对照组,定量和定性测定两组的神经生长因子活性,对比蛇毒纤溶酶和神经生长因子的氨基酸序列.结果蛇毒纤溶酶在20U/ml时具有明显的神经生长因子活性,N-末端氨基酸序列与神经生长因子同源性达80～90%.结论蛇毒纤溶酶具有明显的神经生长因子活性,其结构与神经生长因子相似.     

赤爱胜蚯蚓(Eisenib Foetide Sorigng)纤溶酶的研究 1.提取纯化和生物化学性质

采用35％和65％的饱和硫酸铵分级沉淀法,获得蚯蚓纤溶酶活性蛋白,并对其纤溶酶的分子量,纤溶酶对其底物专一性,温度对纤溶酶的影响,纤溶酶最适pH值和pH值稳定性,纤溶酶的等电点,抑制剂对蚓蚯纤溶酶的影响几方面的测定,从而确定了蚯蚓纤溶酶的生物化学性质。     

蚯蚓纤溶酶组分及其基因的多态性

生化制备证明,蚯蚓纤溶酶为一组含有10个以上同工酶组分的混合酶.为了研究这些组分的DNA和蛋白质序列的异同,本文通过对数据库中已报道的28条蚯蚓纤溶酶基因按相似性进行归类,将它们分为4组(基因型).同一组中的序列具有共同特征,即保守的N-末端、C-末端和相同的结构域,而且这些结构域在序列中分布的位置也相同;但它们之间在中间部分存在明显差异,这些差异说明了基因型中存在多态性.这种多态性可能是它们在体内溶栓的药理药效作用存在差异的结构基础.     

蚓激酶的克隆及其对BHK细胞的作用

蚯蚓具有活血化瘀的功效 ,在我国作为中药 (地龙 )使用已经有上千年的历史 .近年来发现蚯蚓体内存在溶血栓成分 ,称为蚓激酶 (ｌｕｍｂｒｕｋｉｎａｓｅ)或蚯蚓纤溶酶 (ｔｈｒｏｍｂｏｌｙｔｉｃｅｎｚｙｍｅｉｎｅａｒｔｈｗｏｒｍ) .自Ｎ .Ｎａｋａ ｊｉｍａ报道从蚯蚓中分离到蚓激酶以来[1] ,国内外已经有许多报道[2～ 6] ,主要侧重于天然提取物的生化研究 .本文通过ＲＴ ＰＣＲ从蚯蚓 (Ｌ .ｂｉｍａｓｔｕｓ)体内获得蚓激酶基因 ,进行表达并观察其对ＢＨＫ细胞的作用 .1　材料与方法1 1　材料正蚓科双胸蚓属蚯蚓 ;反转录试剂 ,质粒ｐｃ…     

Cloning of thrombolytic enzyme (lumbrokinase) from earthworm and its expression in the yeast Pichia pastoris

Lumbrokinase (PI239, GenBank Accession No. AF433650) from the earthworm Lumbricus bimastus has been identified. The cDNA of PI239 is composed of 852bp and includes an open reading frame that encodes two parts of the protein: a signal peptide of 44 amino acids and a mature peptide of 239 residues. The cDNA of PI239 exhibits a high degree of sequence identity with other lumbrokinase genes, ranging from 87.6% (F-III-I) to 98.3% (EFE-3). The gene encoding the native form of PI239, with a 5' non-functional end removed, was obtained by PCR amplification and was sub-cloned into pPICZalpha-A, a yeast expression and secretion vector. SDS-PAGE and Western blot analyses showed that rPI239 secreted into the culture medium was specifically recognized by the wild type lumbrokinase polyclonal antibody and was able to dissolve artificial fibrin plates.     

Isolation, partial purification, and characterization of the cytochrome P-450-dependent monooxygenase system from the midgut of the earthworm Lumbricus terrestris.

1. Cytochrome P-450 was purified from microsomes of the midgut of the earthworm Lumbricus terrestris up to a maximal specific content of 5.5 nmol P-450/mg protein. 2. At least 3 different cytochromes P-450 with apparent molecular weights of 48,000, 51,000 and 53,000 were identified by SDS-PAGE. 3. Western blot analysis with various polyclonal antibodies did not show structural epitopes common to the cytochromes P-450 of rodents or yeast and L. terrestris. 4. The microsomes contained about 43 pmol P-450/mg protein corresponding to 0.51 nmol P-450/g midgut and 64 pmol P-450/g body weight, respectively, and converted benzyloxyresorufin into resorufin with a Vmax of 2.12 pmol resorufin/min.mg protein and a Km of 770 nM benzyloxyresorufin at 25 degrees C, pH 8.0. 5. The microsomes exhibited a NADPH-cytochrome P-450 reductase activity of 9.4 nmol cytochrome c/min.mg protein. 6. The apparent molecular weight of the threefold-purified reductase was 63,000.     

Isolation,partial purification,and characterization of the cytochrome P-450-dependent monooxygenase system from the midgut of the earthworm Lumbricus terrestris

1. Cytochrome P-450 was purified from microsomes of the midgut of the earthworm Lumbricus terrestris up to a maximal specific content of 5.5 nmol P-450/mg protein.2. At least 3 different cytochromes P-450 with apparent molecular weights of 48,000, 51,000 and 53,000 were identified by SDS-PAGE.3. Western blot analysis with various polyclonal antibodies did not show structural epitopes common to the cytochromes P-450 of rodents or yeast and L. terrestris.4. The microsomes contained about 43 pmol P-450/mg protein corresponding to 0.51 nmol P-450/g midgut and 64 pmol P-450/g body weight, respectively, and converted benzyloxyresorufin into resorufin with a V_max, of 2.12 pmol resorufin/min.mg protein and a K_m of 770 nM benzyloxyresorufin at 25°C, pH 8.O.5. The microsomes exhibited a NADPH-cytochrome P-450 reductase activity of 9.4 nmol cytochrome c/min.mg protein.6. The apparent molecular weight of the threefold-purified reductase was 63,000.     

Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noninducibility of cytochrome P-450 in the earthworm Dendrobaena veneta

Cytochrome P-450 has been measured in the earthworm Dendrobaena veneta (Rosa) in a direct spectrophotometric procedure. The P-450 was found not in the dense microsomal fraction, but in the less dense overlying fraction often referred to as buffy coat. Earthworm P-450 was not induced by 3-methylcholanthrene or phenobarbitol.     

The immunologically protective P-4 antigen of Leishmania amastigotes. A developmentally regulated single strand-specific nuclease associated with the endoplasmic reticulum

The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the P1 zinc-dependent nuclease of Penicillium citrinum (20.8%) and the C-terminal domain of the 3' nucleotidase of Leishmania donovani (33.7%). Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP > CMP. Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and single-stranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.     

Expression and characterization of earthworm Eisenia foetida lumbrokinase-3 in Pichia pastoris

Lumbrokinase-3 (LK-3, AY438622), first cloned from the earthworm Eisenia foetida in our laboratory, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the LK-3 gene was sub-cloned into yeast pPIC9K expression vector and transformed into the Pichia pastoris GS115 cells by electroporation. High level expression of LK-3 in yeast cells was confirmed with a different induction time. The activity of expressed LK-3 was observed in fibrin plates. In addition, the expressed LK-3 protein could dissolve fibrinogen and bovine serum albumin. The use of this system for the high level production of biological protein is implicated from this study.