羊草cDNA文库的构建及果聚糖水解酶的分离与鉴定

羊草(Leymus chinensis(Trin.) Tzvel.)又称碱草，隶属禾本科赖草属，是欧亚大陆草原区东部草甸草原及干旱草原上的重要建群种之一。作为一种兼具重要经济价值和生态价值的优良牧草，羊草受到了广泛的关注。但长期以来，对羊草的研究主要集中在生态学、生殖生物学方面，在分子生物学方面知之甚少。为了保存羊草基因资源，并在基因水平上研究羊草生物代谢的调控机理，本研究采用羊草根、叶片混合后做为材料，构建cDNA文库，并对文库中部分基因序列进行了分析。同时从中克隆获得羊草果聚糖水解酶全长基因并对这些基因进行了深入的生物信息学分析、转化毕赤酵母研究初步确定其功能，为深入了解羊草代谢的分子机制提供理论依据。主要结果如下： 1. 成功地构建了羊草根、叶混合cDNA文库，原始文库滴度达到4×106 pfu/ml，扩增文库滴度接近1011 pfu/ml ，重组率达97% 。PCR检测插入片段，均在0.5 kb到3 kb之间，l kb以上占68%。从文库中检测到了TC、γ-TMT、FEH基因，文库覆盖度达到要求且为PCR筛选文库提供了可能。 2. 随机挑取经检测过的597个单克隆进行测序，去除插入片段小于和污染序列后，获得了584条高质量的序列。所有584条EST序列与NCBI的核酸数据库比对时，有30.99%的EST序列与己知序列有很大的同源性：而与蛋白质数据库进行比对时，有61.27%的EST序列与已知序列有很大的同源性。核酸比对中，有32.87%的序列为未知功能新基因，而蛋白质比对结果只有11.27%的序列为未知功能新基因。其中获得5条全长基因。 3. 文库中测序得到果聚糖水解酶（FEH）片段，依据其核酸、蛋白序列，以羊草根茎为材料，结合果聚糖水解酶基因的保守序列设计引物，通过 RACE 方法，获得羊草果聚糖水解酶基因 Lc 1-FEH 的全长序列（2040bp），包含一个 1803bp 的开放阅读框，采用生物信息学方法对该基因编码蛋白质进行功能分析，该基因编码的氨基酸序列具有明显的果聚糖水解酶类蛋白特征(NDPNG，FRDP 和[WEC (V/P)D] 结构域)，其分子量为 66.8kD，等电点 pI 为 5.49，是一种酸性蛋白质。同源性分析结果表明Lc1-FEH与单子叶植物小麦、大麦和黑麦草细胞壁类酵素酶同源性最高，分别为89％、87％ 和72％。运用实时定量方法对Lc 1-FEH表达量在羊草发育各时期及不同逆境处理下进行测定，结果发现，幼苗中以叶中表达量最低，根茎中表达较高，成苗中花梗中的表达量最高；Lc1-FEH在转录水平明显受碱、ABA、SA及低温胁迫诱导，随着胁迫时间延长，表达量迅速增加，到达到最大值，之后表达水平逐渐降低，在盐、干旱的诱导下的表达量迅速降低。 4. 羊草果聚糖水解酶Lc1-FEH基因在毕赤酵母中的高效表达 将pMD-Lc1-FEH 质粒经双酶切后构建果聚糖酵母表达载体 pPICZα- Lc1-FEH 。将重组表达载体线性化后电击转化毕赤酵母Pichia pastoris X33，经抗生素 Zeocin 和酵母 PCR 筛选获得高效表达酵母工程菌。毕赤酵母表达的果聚糖水解酶蛋白经SDS-PAGE 分析表明Lc1-FEH表观分子量为 67 kD 左右。其最适反应 pH 值为 5.5，在 pH 值为 4.5~6.5 的范围内能保持较高的酶活力；表达Lc1-FEH的最适反应温度为 30 ℃；在温度在 20～30℃度范围内有较高的酶活。 Lc 1-FEH能够水解含有β-2，1糖苷键类型果聚糖：蔗果四糖、菊粉、6-蔗果三糖；而对β-2，6糖苷键类型果聚糖：6-蔗果三糖、新蔗果三糖、细菌类果聚糖及蔗糖基本不具水解活性。

酵母拮抗菌和外源水杨酸对核果类果实抗病相关基因的诱导表达

在果实采后贮藏过程中，病原真菌的侵染会引起果实腐烂，造成巨大的经济损失。利用生物和非生物因子诱导果实抗病性，已经成为采后病害防治领域的一个研究热点。本文主要利用RT-PCR和RACE技术克隆果实抗病相关基因，通过分子杂交和蛋白羰基化免疫检测技术，研究了外源SA和酵母拮抗菌诱导果实抗病性机理，结果表明： 1. 通过优化RNA提取方法，能从含有多糖的冬枣、葡萄、甜樱桃、桃、番茄等果实中提取到质量较好的RNA，用于RT-PCR和Northern杂交。 2. 采用RT-PCR和RACE方法，从甜樱桃果实克隆了两个抗氧化相关基因CAT2（Genbank：EF165590）和GPX（Genbank：EF165591）和两个PR基因GLU-1（Genbank：EF177487）和GLU-3（Genbank：EF177488)。其中CAT2全长cDNA序列为1479 bp，编码492个氨基酸；GPX全长cDNA序列为513 bp，编码170个氨基酸；GLU-1全长cDNA序列为1050 bp，编码349个氨基酸；GLU-3部分cDNA序列为454 bp，编码141个氨基酸。 3. 酵母拮抗菌Pichia membranaefaciens处理不同成熟度的甜樱桃果实，能显著降低果实贮藏期间青霉病（Penicillium expansum）的发生，并且对低成熟度果实的病害防治效果更为明显。酵母拮抗菌的抑病机理与减轻了甜樱桃果实蛋白羰基化程度，诱导了果实抗氧化酶基因（CAT和GPX）和PR基因（GLU-1）的表达和提高了抗氧化酶（CAT和GPX）和β-1,3-葡聚糖酶的活性有关。 4. 四种酵母拮抗菌P. membranaefaciens, Cryptococcus laurentii, Candida guilliermondii和Rhodotorula glutinis处理桃果实，可显著降低贮藏期间的褐腐病（Monilinia fructicola）。这是由于酵母拮抗菌能抑制病原菌侵染造成的氧化胁迫和蛋白羰基化。此外，酵母拮抗菌处理还能显著诱导CAT、POD、几丁质酶、β-1,3-葡聚糖酶活性及相应基因的表达。 5. 水杨酸（SA，2 mM）处理采后不同成熟度的甜樱桃果实，能显著降低青霉病的危害。其抑病机理与SA处理能减轻P. expansum侵染引起的果实蛋白羰基化程度，显著提高CAT、GPX和β-1,3-葡聚糖酶基因的表达和相关的酶的活性有关。而2 mM的SA处理对P. expansum的生长没有直接抑制作用。 6. 水杨酸（SA，2 mM）与P. membranaefaciens（1×108 CFU/ml）配合处理能显著降低低温贮藏期间桃果实的褐腐病，并能提高几丁质酶、β-1,3-葡聚糖酶和POD的活性和相关基因的表达。另外，2 mM的SA对拮抗菌P. membranaefaciens的生长没有影响，但能够抑制病原菌M. fructicola的孢子萌发和菌丝扩展。

水稻2,3-氧化鲨烯环化酶基因的功能研究

植物通过异戊二烯代谢途径合成多种具有生物活性和功能的三萜及甾醇类化合物，它们在调节植物生长发育、维持膜的完整和功能、抵抗病原微生物侵染中发挥着重要的作用。2,3-氧化鲨烯为三萜和甾醇合成途径的分枝点，参与这一关键步骤的酶被通称为2,3-氧化鲨烯环化酶（OSCs）。本研究系统分了水稻基因组中全部11个OSC基因序列，发现其中四个可能为假基因。亚种间非同义替换率Ka和同义替换率Ks的比值（Ka/Ks）以及进化树的分析表明OsOSC8是单子叶植物特有的功能保守基因，而OsOSC9在水稻两个亚种间发生了功能快速进化，这种快速进化的基因往往参与植物和病原菌相互作用的代谢途径。 根据基因结构、表达谱以及与其它植物已知功能的OSC酶氨基酸序列的比对推测OsOSC3可能具有环阿屯醇合成酶的功能，参与植物甾醇的合成，而OsOSC7、OsOSC10和OsOSC11可能具有β-香树素合成酶的功能，其余OSCs可能参与合成其它三萜化合物。为了进一步分析和验证OSCs酶的功能，将水稻7个OSC基因的开放阅读框（ORF）构建到酵母表达载体并在pichia酵母中表达，发现仅有OsOSC9和OsOSC12能够将酵母内源的2,3-氧化鲨烯分别环化为四环三萜化合物Parkeol和植物中稀有的五环三萜化合物Isoarborinol，目前还未在其它植物中发现参与这两种三萜化合物的基因。另外，水稻所有的OSC基因均不能互补酵母羊毛甾醇缺陷型菌株，表明水稻OSCs不具有合成羊毛甾醇的功能。 RNAi沉默以及启动子融合GUS的表达实验发现OsOSC8可能参与花粉的发育，该基因的下调影响水稻的育性，暗示水稻中存在一个可能与雄性不育有关的三萜代谢途径。水稻其它OSC基因RNAi植株可能在逆境环境和病原菌侵染下才会显现出表型。

Expression, purification and characterization of recombinant Jerdonitin, a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC50 of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin. (C) 2009 Elsevier Ltd. All rights reserved.

甜蛋白Monellin在毕赤酵母中的分泌表达

在西非热带植物Dioscoreophyllum cumminsii中存在着一种由两条多肽构成的甜度极高的蛋白Monellin。本研究根据已知的Monellin晶体衍射分析结果和毕赤酵母(Pichia pastoris)基因偏爱密码子设计并合成了连接两条多肽的重组基因,插入到毕赤酵母分泌表达质粒pGAPZαA中。扩增后,电击穿孔转化毕赤酵母GS115,筛选高产菌株放大培养。以5升罐发酵培养,表达量为150mg/L。经纯化,可以获得大于130mg/L的具有高甜度的活性蛋白。

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

碱性成纤维细胞生长因子的克隆、表达、纯化、生物活性及与小分子的相互作用

人碱性成纤维细胞生长因子（bFGF）是一种单链的具有与肝素结合能力的多肤分子，它能够调节内皮细胞及其它来源于中胚层和神经外胚层等多种细胞的生长、分化、迁移和凋亡，同时是一种重要的血管生成因子，因此具有广泛的临床应用价值。本论文就将具有多种生物学功能的碱性碱纤维细胞生长因子开发为基因工程药物所存在的一些基本问题，进行了大量细致的研究工作并获得一些具有新意的研究结果。应用现代基因工程技术分别在原核E.coli系统和真核Pichia pastoris系统件1克隆表达了人碱性碱纤维细胞生长因子，生物活性测定结果表明在毕赤酵母体系中表达的重组人碱性碱纤维细胞生长因子的活性高于E.coli中表达的bFGF的活性。筛选出高分泌型的Pichia pastoris工程菌，并建立了高效表达的方法；经过一步亲和层析既获得了电泳纯的bFGF其产量为100mg／L发酵液。SDS-PAGE电泳以及Western blOt实验都证明了其具有天然bFGF的性质，并用紫外、荧光光谱刘于毕赤酵母表达体系表达的重组人bFGF的三维结构进行了表征，用圆二色谱对其二级结构进行了表征，同时用质谱测定了其精确分子量为17931 Da．首次研究了不同价态的稀土离子与碱性碱纤维细胞生长因子相互作用引起bFGF不同的构象及活性变化，以及聚金属氧酸盐（POM）在低浓度能够刺激bFGF的活性，在高浓度抑制bFGF活性的现象。

均质分析生物器件的研究

以蛋白质为基础的分析生物器件，如传感器和生物芯片等，为人们提供了有效的分析技术平台。而蛋白质固定的均质性则是评估分析生物器件质量的一个主要指标。因此，本实验以两种蛋白质为模式蛋白研究蛋白质固定的均质性问题。通过基因操纵构建融合蛋白Protein-Linker-Cysteine。在此设计中，半胱氨酸提供的自由疏基能够在金表面形成Au一S键，在琉基修饰的玻片表面形成-S-S-键实现蛋白质的均质定向固定；Linker可减少基因修饰对蛋白质折叠的影响。构建表达载体pPIC-GOxm（GOx-Linker-Cysteine），利用原生质体转化法将其转进毕氏酵母Pichia Pastoris，采用QSepharoseTM FastFlow阴离子交换柱纯化融合蛋白。动力学性质分析表明GOxm具有与野生型葡萄糖氧化酶相类似的Km和Kcat值，电化学实验结果显示Goxm传感器具有较高的响应电流；GOxm传感器具有较好的互换性，其相对误差为9.48%，GOxw（wild type GOx）相对误差为19.98％，而传统传感器的相对误差为17.54%。原子力显微镜图像显示融合蛋白GOxm能够利用金表面的HC尸位点形成类似六边型晶格的自组装单分子层，而野生型GOxw在金表面为非特异性吸附，形成多层固定导致分子间的聚集。通过利用-S-S-和非特异性吸附，分别制成GOxm蛋白芯片和Goxw蛋白芯片。酶学显色后，通过光学信号评估芯片的均质性，结果表明Goxm能够利用-S-S-形成均质定向固定，10次重复的变异系数小于60k，而GOxw则不能形成均质固定，点阵间的变异系数变化幅度非常大，从40％到80％。构建表达载体pET-BLC，pET-BL。将其转化进大肠杆菌AD494中。原子力显微镜研究整合有磷脂和经抽提去掉磷脂的蛋白在金表面的固定。原子力显微镜图像显示融合蛋白BLC能够利用Au-S键在金表面形成均匀固定，而野生型蛋白在金表面不能形成均匀的固定。蛋白质在金表面的固定受金表面拓扑结构和磷脂的影响。以上的实验结果表明通过此种固定方法可改善分析生物器件的均质性，提高其质量。

无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因表达载体的构建及其在毕赤酵母中的表达

研究背景与目的：近二十年来，抗生素的广泛使用以及一些不当应用导致临床上出现大量的耐药性病原菌，所以不易产生耐药性的抗菌肽就成为目前研究的热点。本课题组此前的研究表明无指盘臭蛙（Odorrana grahami）皮肤抗菌肽具有广谱抗菌活性，但对真核细胞没有毒性，因此有成为新型药物的潜力。本研究采用毕赤酵母真核表达系统来生物合成抗菌肽Odorgrin A和Odorgrin C，为大量获取抗菌肽资源提供技术支撑。 方法：依照Odorgrin A和C的氨基酸序列、采用酵母偏爱密码子分别设计并化学合成了相应的目的基因序列。目的片段从合成质粒上用Xho Ι和EcoR Ι双酶切下后，与经同样限制酶完全酶切pPIC9K载体所获得的两个大片段直接连接，并转化至大肠杆菌DH5α。用PCR扩增、酶切及测序检测，鉴定正确的重组质粒。提取大量表达载体pPIC9K - Odo A和C并使之线性化后经电击法分别转化毕赤酵母（Pichia pastoris）GS115宿主菌，用营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测，鉴定并筛选出对G418具高抗性的Odorgrin A和C重组酵母菌。用甲醇对之进行诱导表达，SDS - PAGE电泳及反相层析检测表达产物，并做抑菌活性检测。 成果：PCR扩增、酶切及测序等结果表明表达载体pPIC9K - Odo A和C构建成功。营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序等证实pPIC9K - Odo A和C已整合入酵母基因组中。SDS - PAGE电泳及反相层析结果表明抗菌肽Odorgrin A和C成功地获得了分泌表达。而抑菌活性实验则检测到部分阳性克隆菌诱导分泌表达的抗菌肽Odorgrin A和C都对测试菌的生长具有较高（>94%）的抑制率。 结论：无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因的表达载体都构建成功，并且都在毕赤酵母系统中获得了成功表达。 Background ＆ Objective: In the recent twenty years, a lot of pathogenic bacteria have come forth in clinic with durable trait derived from making use of and abusing the traditional antibiotics. Therefore, studying antimicrobial peptides, not be easy to be invalidated by durable bacteria, are becomimg popular and important. The skin antimicrobial peptides of Odorrana grahami with broad spectrum antibacterial activity and no toxicity to eukaryotic cell, discovered by previous research work of our workgroup, are looked forward to being potential medication. Pichia pastoris expressional system was used for biosynthesis antimicrobial peptides Odorgrin A and Odorgrin C in this study, for producing abundant antimicrobial peptides. Methods: The foreign fragments which included Odorgrin A or Odorgrin C gene according to their amino acid sequence respectively were synthesized based on the biased codon usage of yeast. The DNA fragments, obtained from the plasmids containing them by digested with Xho Ι and EcoR Ι, were directly ligated with the two bigger fragments obtained from the vector pPIC9K by digested with the same restriction enzymes. And then they were transformed into Escherichia coli DH5α to be selected and amplified positive colonies. The recombinants were testified by using PCR amplification, enzymes digestion and sequencing of the foreign fragment. After the expressional vector pPIC9K - Odo A and pPIC9K - Odo C were linearized, they were transformed into Pichia pastoris GS115 strain by the electroporation. Then the positive colonies which were of the highest geneticin resistant were selected through auxotrophic screening, genetic resistant screening, PCR amplification and sequencing of the inserted fragment. Methanol was used to induce the recombinant yeasts to express the foreign gene. SDS-PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment were used to testify the expressional products. Results: The evidences of PCR, enzymes digestion and sequence analysis confirmed that the expressional vector pPIC9K - Odo A and pPIC9K - Odo C have been constructed correctly. The results of auxotrophic screening, of genetic resistant screening, of PCR and sequencing of the foreign fragment showed that Odorgrin A and Odorgrin C gene have been homologous integrated with the Pichia pastoris genome. And it was also testified that antimicrobial peptides Odorgrin A and Odorgrin C have been expressed successfully by using SDS - PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment. Conclusion: The expressional vector of the skin antimicrobial peptides Odorgrin A and Odorgrin C gene of Odorrana grahami have been constructed correctly and both of the genes have been expressed successfully in Pichia pastoris system in this study.

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis

P>NF-kappa B is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-kappa B-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor kappa B (I kappa B)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-kappa Bs and phylogenetic analysis suggested that EsRelish was a member of the NF-kappa B family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking, The full-length DNA of the scallop H2A was 696 bp long, including a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 228 bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated. (C) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein

Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603 bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92 kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri. (c) 2006 Elsevier Ltd. All rights reserved.

CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coil, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.