Molecular cloning, expression of a peroxiredoxin gene in Chinese shrimp Fenneropenaeus chinensis and the antioxidant activity of its recombinant protein

Peroxiredoxin (Prx) is known to be an antioxidant protein that protects the organisms against various oxidative stresses and functions in intracellular signal transduction. A Prx gene was firstly isolated in the crustacean, Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA consists of 942 bp with a 594 bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22041.17 Da with an estimated pI of 5.17. Sequence comparison showed that Prx of F. chinensis shares 76%, 73% and 72% identity with that of Aedes aegypti, Branchiostoma belcheri tsingtaunese and Drosophila melanogaster, respectively. Northern blot analysis revealed the presence of Prx transcripts of F chinensis in all tissues examined. Real-time PCR analysis indicated that the Prx showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with Vibrio anguillarum. In addition, a fusion protein containing Prx was produced in vitro. LC-ESI-MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. And the purified recombinant proteins were shown to reduce H2O2 in the presence of dithiothreitol. (c) 2007 Elsevier Ltd. All rights reserved.

固氮蓝藻与非固氮蓝藻谷氨酰胺酶的基因克隆、表达及其特性分析

蓝藻是唯一可以进行有氧光合作用的原核生物，是水生食物链主要的初级生产者。氮素是蓝藻细胞必需的大量营养元素之一，揭示蓝藻如何应对环境中氮素的变化、维持自身碳氮平衡的分子机理，对深刻理解蓝藻与环境的相互作用、有效促进或控制蓝藻的生长与繁殖，有重要的理论和实践意义。已有的研究发现，蓝藻细胞的碳氮平衡主要是通过调控氨同化途径中的关键酶类实现。但先前的研究主要集中在固氮蓝藻谷氨酰胺合成酶（GS）-谷氨酸合成酶(GOGAT)循环的特性分析方面，而对催化谷氨酰胺水解生成谷氨酸和氨的主要酶之一谷氨酰胺酶的报道极少，其分子特性及生理学意义尚不明了。因此，本论文以模式固氮蓝藻鱼腥藻7120 和非固氮蓝藻集胞藻6803 为材料，采用分子生物学和生物化学方法，对蓝藻谷氨酰胺酶进行体外研究，并对其生物学功能进行了初步探讨，获得了如下主要结果：1）对体外重组蛋白的酶活性检测发现，两类蓝藻基因组编码的假定性谷氨酰胺酶，均具有谷氨酰胺酶催化活性，表明基因组注释是准确的；2）固氮蓝藻重组酶（All2934、All4774）与非固氮蓝藻重组酶（Slr2079）酶学特征差异显著，具有不同的最适pH、温度及底物亲和力；3）固氮蓝藻重组酶All2934 催化活性受磷酸盐的激活，而非固氮蓝藻重组酶Slr2079 在高Na+浓度下活性更高；4）RT-PCR 分析结果表明，在正常培养条件下，两类蓝藻的谷氨酰胺酶基因在细胞内均有表达；5）在缺氮培养条件下，固氮蓝藻谷氨酰胺酶基因all2934 的表达水平发生明显变化，而all4774 保持相对稳定，表明前者可能在这类细胞应对氮饥饿过程中起重要作用；6）在正常培养条件下，非固氮蓝藻谷氨酰胺酶基因的缺失突变体(Δslr2079)与野生型表型相似，但在盐胁迫条件下，突变体生长速率及光合放氧活性均高于野生型，表明该基因可能在提高非固氮蓝藻细胞高盐耐受力方面起负调控作用。上述重要发现，不仅初步揭示了光合自氧生物谷氨酰胺酶体外重组酶的分子特征，也为进一步研究谷氨酰胺酶在蓝藻细胞内的专一性功能奠定了重要基础。

Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris

The lipase genes of Yarrowia lipolytica, LIPY7 and LIPY8, fused with FLO-flocculation domain sequence from Saccharomyces cerevisiae at their N-termini, were expressed in Pichia pastoris KM71. Following the induction with methanol, the recombinant proteins were displayed on the cell surface of P. pastoris, as confirmed by the confocal laser scanning microscopy. The LipY7p and LipY8p were anchored on P. pastoris via the flocculation functional domain of Flo 1 p. The surface-displayed lipases were characterized for their application as the whole-cell biocatalyst. These lipases can also be cleaved off from their anchor by enterokinase treatment to yield functionally active proteins in the supernatant offering an alternative purification method for LipY7p and LipY8p. (c) 2007 Elsevier Inc. All rights reserved.

Evaluation of different culture conditions for high-level soluble expression of human cyclin A(2) with pET vector in BL21 (DE3) and spectroscopic characterization of its inclusion body structure

In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A, in Escherichia coli BL21(DE3), and demonstrated that the highest protein yield was obtained using TB(no glycerol) + 0.5% glucose medium at 25 degrees C. By single immobilized metal ion affinity chromatography, we got highly purified human cyclin A(2) with a yield ranged from 20 to 30 mg/L. By amyloid-diagnostic dye ThT binding and Fourier transform infrared spectroscopy, we observed a significant decrease in alpha-helix content and an increase in beta-sheet structure in cyclin A(2) inclusion body in comparison to its native protein, and confirmed the resemblance of the internal organization of cyclin A(2) inclusion body and amyloid fibrils.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Two thymosin-repeated molecules with structural and functional diversity coexist in Chinese mitten crab Eriocheir sinensis

Recently, beta-thymosin-like proteins with multiple thymosin domains (defined as thymosin-repeated proteins) have been identified from invertebrate. In the present study, the cDNAs of two thymosin-repeated proteins (designated EsTRP1 and EsTRP2) were cloned from Chinese mitten crab by expressed sequence tags (EST) techniques. BLAST analysis presented three and two thymosin domains in EsTRP1 and EsTRP2, respectively, with the identities amongst the five domains varying from 47% to 100%. Both EsTRP1 and EsTRP2 shared high similarities with previously identified vertebrate beta-thynnosins and invertebrate thymosin-repeated proteins (TRPs) with the identities ranging from 43% to 78%, indicating that EsTRPs were new members of the beta-thymosin family. Real-time RT-PCR assay was adopted to determine the tissue distribution of EsTRPs and their temporal expression profile in hemocytes after pathogen stimulation and injury challenge. The expression of EsTRP1 transcript was predominantly detectable in the tissues of hemocytes, hepatopancreas and gonad with the highest expression in hemocytes, while the highest expression level of EsTRP2 was found in heart. EsTRP1 mRNA expression in hemocytes significantly increased at 3 and 48 h after Listonella anguillarum challenge, but there was no significant variation in EsTRP2 temporal expression profile. The injury challenge reduced the mRNA expression of EsTRPs, with the down-regulation of EsTRP2 expression occurred earlier than that of EsTRP1. The cDNA fragments encoding their mature peptides of EsTRP1 and EsTRP2 were recombined and expressed in Escherichia coli. The activities of recombinant proteins (rEsTRP1 and rEsTRP2) were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and lysoplate assay. rEsTRP2 could significantly accelerate the growth of human hepatocellular carcinoma cell line, but there was no significant effect of rEsTRP1 on the tumor cell proliferation. Both rEsTRP1 and rEsTRP2 did not possess the ability of killing Micrococcus luteus and L. anguillarum. The differences in the tissue distribution of mRNA transcripts, the response to pathogen stimulation and injury challenge, and the effect of recombinant proteins on human cell proliferation, indicated that there were functional diversity between the two structurally different molecules, EsTRP1 and EsTRP2. (C) 2009 Elsevier Ltd. All rights reserved.

Identification, expression, function and localization of a DUF985 domain-containing hypothetical gene from amphioxus Branchiostoma belcheri

The progress in genome sequencing has led to an increasing submission of uncharacterized hypothetical genes with the domain of unknown function, DUF985, in GenBank, and none of these genes is related to a known protein. We therefore underwent an experimental study to identify the function of a DUF985 domain-containing hypothetical gene BbDUF985 (GenBank Accession No. AY273818) isolated from amphioxus Branchiostoma belcheri (B. belcheri). BbDUF985 was successfully expressed in both prokaryotic and eukaryotic systems, and its recombinant proteins expressed in both systems definitely exhibited an activity of phosphoglucose isomerase (PGI). Both tissue-section in situ hybridization and immunohistochemistry demonstrated that BbDUF985 was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In CHO cells transfected with the expression plasmid pEGFP-N1/BbDUF985, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that BbDUF985 is a cytosolic protein. In contrast, Western blotting indicated that BbDUF985 was also present in amphioxus humoral fluids, suggesting that it exists as a secreted protein as well. Our study provided a framework for further understanding the biochemical properties and physiological function of DUF985-containing hypothetical proteins in other species. (c) 2008 Elsevier Inc. All rights reserved.

中国明对虾免疫系统中抗氧化相关基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明，当对虾等甲壳动物受到外界病原刺激时，其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢，引起呼吸爆发，产生多种活性氧分子。另外，受到病原侵染的对虾还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物，但同时由于活性氧分子反应的非特异性，它们也会对宿主的细胞、组织和器官造成严重伤害，进而导致对虾生理机能的损伤和免疫系统的破坏。所以，消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力，提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶（mMnSOD）、胞质型超氧化物歧化酶（cMnSOD）、过氧化氢酶（Catalase）和过氧化物还原酶（Peroxiredoxin）等四种与免疫系统相关的抗氧化酶基因，分析了它们的分子结构特征，组织分布及应答不同病原刺激的表达变化模式，并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶（SOD）基因，通过序列比对分析发现，其中一个为mMnSOD基因，另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基，其中开放阅读框为660个碱基，编码220个氨基酸，其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明，该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示，对虾感染病毒3 h时，该基因在血细胞和肝胰脏中的转录水平显著升高。此外，通过构建原核表达载体，本研究对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基，其中开放阅读框为861个碱基，编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示，cMnSOD基因在对虾被检测的各个组织中均有表达。 另外，半定量RT-PCR结果表明，对虾感染病毒23h时，该基因在肝胰脏中的转录上升到正常水平的3.5倍；而感染后59 h时，该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物，结合RACE技术，从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段，片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现，该基因在肝胰脏、鳃、肠和血细胞中表达水平较高，在卵巢、淋巴器官和肌肉中的表达水平相对较弱；感染病毒23 h和37 h时，对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息，结合SMART-RACE技术，从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因（Peroxiredoxin）， 该基因的cDNA全长为942个碱基，其中开放阅读框为594个碱基，编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da，pI为5.17。Northern blot结果表明，Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示，弧菌感染后，该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外，对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明，复性后的重组蛋白能在DTT存在的条件下还原H2O2。

扇贝丝氨酸蛋白酶抑制剂基因CfKZSPI及Aikunitz的克隆与重组表达

扇贝养殖是我国重要的海水养殖产业，然而自1997 年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且严重影响了该产业的健康发展。丝氨酸蛋白酶抑制因子及丝氨酸蛋白酶在无脊椎动物的免疫应答中起着核心作用，它们的协同作用直接导致外界病源入侵的信号转导和级联放大，并进一步激活一系列防御体系，如黑化反应、血液凝结和抗菌肽的合成等。因此，克隆扇贝参与免疫防御的丝氨酸蛋白酶抑制剂基因并对其功能进行研究，将有助于进一步研究扇贝的免疫防御机制，丰富和发展无脊椎动物免疫学的内容。 运用大规模EST技术和RACE技术从栉孔扇贝中克隆出一个Kazal型丝氨酸蛋白酶抑制剂基因，定名为CfKZSPI。该基因cDNA序列全长1788bp，其中5' 非编码区（Untranslated Region, UTR）为97 bp，3' UTR161 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，开放阅读框（Open Reading Frame, ORF）含有1530 bp，编码509 个氨基酸残基。对其推测氨基酸序列进行分析，发现其中包括22个氨基酸残基组成的信号肽序列和12个Kazal型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR（quantitative real time PCR）对鳗弧菌浸泡刺激后栉孔扇贝血淋巴中CfKZSPI 的 mRNA表达量进行了检测，发现其mRNA 的表达量在鳗弧菌刺激后3h明显上升，达到空白组的43.6倍；然后在6h时有所下降，为空白组的15.0倍；随着菌刺激时间的增长，CfKZSPI基因的 mRNA 表达量急剧增加，在刺激后8h，12h，24h分别达到空白组的174.1，207.8，675.4倍。统计分析发现3h（P=0.019<0.05）和12h（P=0.020<0.05）时，CfKZSPI基因mRNA表达量与空白组差异均显著。为了研究栉孔扇贝CfKZSPI的蛋白活性，将其第十二个结构域克隆到pET-32a（+）载体中，转化大肠杆菌Rosetta-gami（DE3）表达菌株，获得可溶性表达的蛋白rCfKZSPI-12，对其进行抑制蛋白酶活性的分析，发现其对胰蛋白酶有很强的抑制活性，而对凝血酶没有抑制活性。当rCfKZSPI-12与胰蛋白酶分子比率为1：1时，约90%的蛋白酶活性被抑制。运用狄更斯作图法研究rCfKZSPI-12对胰蛋白酶的抑制能力，结果发现其对胰蛋白酶的抑制常数为173 nmol L-1。 采用同样方法从海湾扇贝cDNA文库中克隆出一个Kunitz型丝氨酸蛋白酶抑制剂基因，定名为Aikunitz。该基因全长632 bp，其中5' UTR 为105 bp，3' UTR 为 245 bp，有一个典型的多聚腺苷酸信号序列（AATAAA）和一个ploy A 尾巴，ORF 含有282 bp，编码93 个氨基酸残基。推测的氨基酸序列N末端有一个20个氨基酸残基组成的信号肽序列，成熟蛋白包括一个Kunitz型丝氨酸蛋白酶抑制剂结构域。采用QRT-PCR对鳗弧菌和藤黄微球菌感染后海湾扇贝血淋巴中Aikunitz 的mRNA的表达量进行了检测，结果发现其在鳗弧菌刺激后3h到9h持续上升，9h时表达量为PBS对照组的4.49倍（P=0.008<0.05），然后开始下降，在72h时表达量为对照组的0.24倍（P=0.021<0.05）；而在藤黄微球菌刺激后3h到12h其表达量上升，其中6h时为空白组的5.95倍（P=0.0004<0.01）；12h以后迅速下降，其中24h的表达量为对照组的0.38倍（P=0.028<0.05）。将Aikunitz基因编码的成熟蛋白按照重组CfKZSPI-12的方法进行重组表达，并对重组蛋白进行抑制蛋白酶和抑菌活性分析。结果发现其对胰蛋白酶和弹性蛋白酶两种丝氨酸蛋白酶都没有抑制作用。抑菌实验同样发现，重组Aikunitz 对供试的革兰氏阳性菌藤黄微球菌和革兰氏阴性菌鳗弧菌和大肠杆菌都不显示明显抑菌活性。

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with-full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron Microscopy, Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV. (c) 2008 Published by Elsevier Inc.

Rapid molecular weight determination of recombinant hirudin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of recombinant hirudin ( rHV-2) was determined rapidly by matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-TOF-MS). The effects of the three types of matrixes were compared and discussed, alpha-cynao-4-hydroxycinnamic acid was proved to be the best matrix. It showed that MALDI-TOF-MS was superior to the traditional method of molecular weight determination of the biological macromolecules. The mass spectrum data proved that the primary structure of rHV-2 was correct and there was no amino acid deletion, mutation and modification in its expression, refolding and purification.

Genomic cloning, expression and recombinant protein purification of alpha and beta subunits of the allophycocyanin gene (apc) from the cyanobacterium Anacystis nidulans UTEX 625

A genomic fragment encoding alpha(APC) and beta(APC) (i.e., alpha and beta units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP ( maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. alpha(APC) and beta(APC) were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications.

Cloning and recombinant expression of a crustin-like gene from Chinese shrimp, Fenneropenaeus chinensis

Antimicrobial peptides or proteins (AMPs) are proved to be one of the most important humoral factors to resist pathogen infection. As an antimicrobial protein, crustin had been described in invertebrates as a component of the innate immune system. A crustin-like gene (CruFc) was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5'-RACE PCR. The full-length cDNA consists of 523 with 405 bp open reading frame encoding 134 amino acids and the deduced peptide contains a putative signal peptide of 17 amino acids. The sequence also contains a whey-acidic protein (WAP) domain at the C-terminal. Transcripts of CruFc were mainly detected in haemocytes and gill by RT-PCR analysis. In addition, another full-length cDNA named CshFc was also cloned from haemocytes of Chinese shrimp and its inferred amino acid sequence lacks the WAP-type 'four-disulfide core' domain. The fusion proteins containing CruFc and CshFc were, respectively, produced and the antimicrobial assays revealed that the recombinant CruFc could inhibit the growth of grain-positive bacteria in vitro but the recombinant CshFc could not inhibit at the same conditions. The difference of antimicrobial activity between recombinant CruFc and CshFc provides the evidence that the four-disulfide core domain of crustin may play an important role in its biological function. (c) 2006 Elsevier B.V. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Silicon Surface Modification with a Mixed Silanes Layer to Immobilize Proteins for Biosensor with Imaging Ellipsometry

One kind of surface modification method on silicon wafer was presented in this paper. A mixed silanes layer was used to modify silicon surface and rendered the surface medium hydrophobic. The mixed silanes layer contained two kinds of compounds, aminopropyltriethoxysilane (APTES) and methyltriethoxysilane (NITES). A few of APTES molecules in the layer was used to immobilize covalently human immunoglobulin G (IgG) on the silicon surface. The human IgG molecules immobilized covalently on the modified surface could retain their structures well and bind more antibody molecules than that on silicon surface modified with only APTES. This kind of surface modification method effectively improved the sensitivity of the biosensor with imaging ellipsometry.