竹叶青蛇毒凝血酶样酶氨基酸序列报道

利用逆转录与聚合酶链反应相结合的RT-PCR法,扩增出竹叶青蛇毒凝血酶样酶(TSV-TEL1)的cDNA;将扩增的cDNA片段克隆入pGEM-T载体中,经末端终止法测定核苷酸序列,推导出竹叶青蛇毒凝血酶样酶的全序列。竹叶青毒凝血酶样酶由234个氨基酸组成并含有1个位于Asn~(20)的N-型糖基结合位点。竹叶青蛇毒凝血酶样酶序列与其它蛇种来源凝血酶样酶具有较大相似性,其与黄绿烙铁头蛇毒凝血酶样酶序列相似度为84%,与美洲矛头蝮蛇毒凝血酶样酶序列相似度为68%,而与牛凝血酶B链序列相似度仅为25%。

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

无指盘臭蛙蛙皮肽及其基因和在制药中的应用

本发明涉及一种无指盘臭蛙蛙皮肽及其基因和在制药中的应用，属于生物医学领域。该活性多肽是中国两栖类动物无指盘臭蛙基因编码的一种单链多肽，分子量3355.95道尔顿，等电点10.14，多肽全序列一级结构为： Gly Leu Gly Gly Ala Lys Lys Asn Phe Ile Ile Ala Ala Asn Lys Thr Ala Pro Gln Ser Val Lys Lys Thr Phe Ser Cys Lys Leu Tyr Asn Gly。编码无指盘臭蛙蛙皮肽的基因由 731个核苷酸组成，其中编码成熟无指盘臭蛙蛙皮肽为第520-616位核苷酸。人工合成的无指盘臭蛙蛙皮肽具有显著的抑制细菌和真菌生长的作用，可以作为制备病原微生物感染疾病的治疗药物被应用。本发明的无指盘臭蛙蛙皮肽具有结构简单、人工合成方便、抗菌谱系广的有益特点。

无指盘臭蛙抗微生物多肽及其基因和在制药上的应用

本发明涉及一种无指盘臭蛙抗微生物多肽及其基因和在制药上的应用，属于生物医学领域。无指盘臭蛙抗微生物多肽是无指盘臭蛙抗微生物多肽基因编码的一种单链多肽，分子量为4248.27道尔顿，等电点为11.47，多肽全序列一级结构为：Gly Leu Phe Thr Leu Ile Lys Gly Ala Ala Lys Leu Ile Gly Lys Thr Val Pro Lys Lys Gln Ala Arg Leu Gly Met Asn Leu Trp Leu Val Lys Leu Pro Thr Asn Val Lys Thr (GLFTLIKGAAKLIGKTVPKKQARLGMNLWLVKLPTNVKT)。编码无指盘臭蛙抗微生物多肽的基因由343个核苷酸组成，其中编码成熟无指盘臭蛙抗微生物多肽为第123-240位核苷酸。人工合成的无指盘臭蛙抗微生物多肽具有很强的抑制细菌和真菌生长的作用，可以作为制备病原微生物感染疾病的治疗药物被应用。本发明的无指盘臭蛙抗微生物多肽具有抗菌谱系广、抗菌活性强的特点。

N49 phospholipase A(2), a unique subgroup of snake venom group II phospholipase A(2)

A novel phospholipase A(2) (PLA(2)) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C-18 chromatography and designated as TM-N49

细胞分化中的tRNA修饰现象

<正> Kersten和Katze等人的实验表明,真菌、粘菌和脊椎动物癌组织的细胞分化过程中存在着tRNA的核苷酸被修饰的现象。修饰发生在某些tRNA特定的位置上。tRNA~(Asn)、tRNA~(Asp)、tRNA~(His)和tRNA~(Tyr)的反密码子第一位(第34号位)的鸟苷(G)被queosine(Q)代替。Q是鸟嘌呤的嘌呤骨架修饰成一个7-去氮结构,它的化学名称应叫7-(4,5-顺式-二羟-2-环戊烯

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus - Brief report

7The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mul of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mul.

大蹼铃蟾（Bombina maxima）白蛋白的皮肤表达、蛋白酶抑制及细胞调亡诱导活性

两栖类动物的皮肤是其得以生存的重要器官，它担负着许多生理功能，如呼吸、水份调节、温度控制、排泄、繁殖、抵抗微生物、防御天敌等。存在于两栖动物皮肤分泌物中的生物活性成分已成为研究热点。目前，己分离鉴定出许多具有各种生物活性的蛋白质及多肤。通过三步分离纯化过程：DEAE-SephadexA-50离子交换，SephadeX075凝胶过滤，和DEAE-SephadexA-50离子交换层析，我们从大蹼铃蟾（Bombinamaxima）皮肤匀浆物中分离得到纯化的大蹼铃蟾皮肤白蛋白（Bm-A-skin）。SDS-PAGE电泳表明，该蛋白为单链蛋白质，在还原状态下表观分子量为67kDa。非还原状态下至少存在三条带，分子量分别为50，55和110kDa。经N-端氨基酸序列分析，其序列均相同，故该蛋白在SDS存在下有同分异构体及多聚体形式。根据所测得的N端氨基酸序列及内肤序列设计引物，通过筛选已构建的大蹼铃蟾皮肤cDNA文库，获得了编码该蛋白的全长cDNA。经序列分析发现该蛋白由三个保守的血清白蛋白结构域组成，并且同人血清白蛋白及牛血清白蛋白的序列相似度分别为39％和38％。随后，我们从血清中也分离纯化到血清白蛋白（BmA-serum），并通过RT-PCR，从大蹼铃蟾肝脏中扩增得到其全长cDNA序列。对由cDNA序列推导的两个大蹼铃蟾白蛋白的氨基酸序列进行比较，发现二者基本相同，只有两个氨基酸的差异，即BmA-skin的Gly417，Ser569，在BmA-serum中均为Asn。造成这两个氨基酸差异的只有一个碱基的突变，即编码BmA-skinGly417，Ser569密码子的第二位碱基A在BmA-serum中变为G。另外，从肝脏获得的BmA-serum的cDNA3'非翻译区还有8个碱基的插入。经扫描光谱分析，BmA-skin含有大量的血红素b，含量为0．95moFinol蛋白，而BmA-serum中含量较少，为0.05mol／mol蛋白。经schiff试剂染色发现，BmA-skin及BmA-serum均为非糖蛋白质。两者均具有抑制trypsin水解小肚底物的活性，但对其它丝氨酸蛋白酶的活性则无抑制，如thrombin、chyomotryPSin、elastase及substilisin。利用表面等离子共振技术研究BmA-skin及BmA-serum与trysin的相互作用，分别得到它们与trrpsin结合的动力学常数，解离平衡常数KD为两者均通过由一对二硫键cys53-Cys62形成的一个暴露的活性位点环，以1：1分子摩尔比同tryrsin形成稳定的非共价结合的复合物，其反应活性位点为Arg58（P1）-His59（P1'）。利用免疫组织化学方法研究发现，BmA-skin广泛地分布于成年大蹼铃蟾上皮细胞的细胞膜及真皮的疏松结缔组织层。表明其在蛙皮肤的生理功能中发挥重要作用，如水及代谢物质交换，渗透压的维持，皮肤呼吸等。另外，我们还从非洲爪蟾（xenopus勿即is）的血清及皮肤中分离到其68扔a的血清白蛋白，经初步鉴定也具有trtPsin抑制活性，但其抑制机制与B．maxima白蛋白不同，还有待于进一步研究。通过MTT法研究发现，BmA-skin对人T淋巴细胞H9、C8166及hemin处理的红白血病细胞K562具有细胞毒活性。三种细胞经BmA-skin处理72h，CC50A片段化，细胞核形态变化及流式细胞仪分析，结果显示，BmA-skin具有选择性诱导细胞调亡的特性。而BmA-serum对三种细胞均无毒性作用，单独的hemin对三种细胞的，胜也很弱。实验结果表明，BmA-skin结合的血红素b可能对其细胞毒及诱导细胞调亡的活性具有较大的贡献。用Cy3标记的BmA-skin与Hg和C8166细胞保温后，发现其进入细胞内发挥作用，这可能是其诱导细胞调亡机制之一。

Resonance Raman spectroscopic study of cytochrome c mutants

The spectroscopic characteristics of cytochrome c(WT) and its mutants(Y67F and N521) in the low frequency region were studied by Resonance Raman technique. The results show that the replacement of phenylalanine for Tyr 67 in WT had a very slight effect on the hydrogen-bonding and conformation of the amino acid residues around propionic acid side chains of heme group. However, large effects on the hydrogen-bonding of internal water with its surrounding amino acid residues and hydrophobility of the home cavity were observed as Asn 52 was substituted with isoleucine, which resulted in conformational regulations of home group and surrounding amino acid residues.

cDNA cloning, characterization and mRNA expression of a pacifastin light chain gene from the Chinese mitten crab Eriocheir sinensis

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036 bp ESPLC cDNA contained an 831 bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67 bp, and a 3'-UTR of 138 bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2 h and 12 h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E sinensis. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146 bp with a 5' UTR of 103 bp, an unusually long 31 UTR of 1519 bp with a canonical polyadenylation signal sequence AATAAA and a potyA tail, and an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5 kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12 h post-Vibrio infection, then recovered to the original level at 24 h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop. (c) 2007 Published by Elsevier Ltd.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have I I amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site I are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.