Progresses on immune-related genes and proteins of abalones

鲍是世界重要的海水养殖经济贝类,但近年来时常发生的疾病给养鲍业带来了大量的经济损失。目前有关鲍免疫相关基因和蛋白的研究报道较少,而这些功能分子对揭示鲍的免疫机制具有关键作用,文章综述了鲍免疫相关基因和蛋白的研究进展,以期为防治鲍疾病的相关研究提供参考。Abalones, belonging to one of the largest marine gastropod mollusks, are economically important seafood in aquaculture worldwide.In recent years, bacterial epidemic infection has been reported in China and other countries, and mass mortality in abalones causes significant economic losses.Immune-related genes and proteins of abalones are seldom reported.However, these functional molecules may play a key role in resisting diseases and maintaining healthy status and are pivotal for studying immunological mechanisms.Here we summarized the advanced research and progresses in abalone immune-related genes and proteins with the purpose of facilitating future study of these target molecules involved in immunological mechanisms.国家高技术研究发展计划项目(863计划)(编号:2007AA091406)资

Application of biomarkers in marine environment monitoring

总结了国内外近十几年可应用于海洋环境污染监测常见的生物标记物及其主要的检测方法,对各自的应用特点进行了阐述,并展望了其应用的前景。其中对DNA损伤的检测作了较为详细的介绍。This paper sums up the recent-year application of biomarkers in marine environment motoring and the main detection methods of them.The features and the future prospects of these biomarkers are also discussed. The introduction of the DNA damage is the most detailed part in this paper.国家自然科学基金资助项目(A20077023,C40106012

Cloning and sequence analysis of hepcidin-like cDNA Hepc2 from liver of Lateolabrax japonicus

Hepcidin(Hepc)是一类性质独特的抗菌肽,具有广谱的抗革兰氏阳性、阴性细菌和真菌等作用。利用RT-PCR和RACE等技术,从经过多种细菌攻毒的花鲈(Lateolabrax japonicus)肝组织中克隆出Hepc全长cDNA,命名为Hepc2,Genbank登录号为AY604195。Hepc2有581个碱基,其中阅读框有258个碱基,编码86个氨基酸。预测氨基酸序列和白鲈及其他鱼种在相同保守的区域有8个半胱氨酸,相对分子量为9418.55dal。在3’非编码区有225bp,包含终止密码子下游189nt处的多腺苷酸化AATAAA信号和212nt处的polyA信号。预测蛋白的信号肽断裂位点在第24和第25个密码子之间。通过与白鲈(Morone chrysops)、人和其他鱼种来源的Hepc cDNA和蛋白质同源性分析表明,从花鲈肝中分离的Hepc2 cDNA属于Hepc基因家族的新成员。Hepcidin is a unique antimicrobial peptide which exerts broad-spectrum antimicrobial action against Gram-negative and Gram-positive bacteria,as well as fungi.A Hepc-like cDNA was amplified from the liver of Lateolabrax japonicus challenged with a mixed bacteria solution.Using RT-PCR and RACE with a specific primer pair,a full length cDNA sequence Hepc2 of the Hepc-like antimicrobial peptide(GenBank accession number:AY604195) was obtained.Hepc2 cDNA is composed of 581 bases,which contains an ORF of 258 bases,encoding 86 amino acids.The deduced amino acid sequence is conserved between white bass and other fish species,which share eight cysteines at the identical conserved position.The relative molecular weight of the protein is 9418.55dal.The 3′non-coding region is composed of 225bp,with a polyadenylation signal AATAAA sequence appearing at position 189 nt,and poly(A) tail at 212 nt,downstream codon TAA.The signal peptide cleavage site of its deduced protein is presumed between codon 24 and 25.High homologies with Hepc cDNAs and proteins of white bass(Morone chrysops),human and other fish are shown.It indicates that Hepc2 cDNA from Lateolabrax japonicus liver is a new member of the Hepc gene family.福建省重大科技项目(2003I005);; 厦门市高新技术项目(3502Z20021052)资

Current status of mangrove germplasm resources and key techniques for mangrove seedling propagation in China

红树植物种质与种苗生产是所有红树林生态恢复工程的基础.本文根据工程实践并结合已有研究资料,采用聚类分析等方法,对中国红树植物资源现状与苗木繁育关键技术进行初步分析.结果表明:中国红树群落可分为低温广布型、广布型、嗜热广布型和热带分布型4种类型;资源分布可划分为琼东沿海、北部湾沿海、珠江口至粤东沿海、闽南和台湾沿海、闽东至浙南沿海5个区域.其中,北部湾沿海红树林种质资源占全国的75.3%.目前中国红树植物苗木种类开发利用率为52.6%,以胎生红树植物为主.红树林苗木生产应注意繁育方法、种实采集与储存、育苗方式、水分和盐度选择、病虫害防治及越冬防寒措施6个技术环节.结合调查和生产实践,归纳分析了中国现有5种红树林苗圃类型(旱地设施苗圃、红树林滩涂苗圃、光滩苗圃、基围塘苗圃和米草滩涂苗圃)的结构和用途特点,为红树林生态恢复工程的系统集成管理提供参考.Mangrove germplasm and nursery operation are the foundations of all mangrove ecological restoration projects.Based on the existing literatures and our own experiences,and by using cluster analysis and other methods,this paper assessed the current status of the mangrove germplasm resources and the key techniques for mangrove seedlings propagation in China.In China,the mangrove communities could be divided into 4 types,including low temperature tolerant widespread type,widespread type,thermophilic widespread type,and tropical type,and the mangrove distribution sites could be divided into 5 regions,i.e.,eastern Hainan coast,Beibuwan Gulf coast,Pearl River estuary and eastern Guangdong coast,southern Fujian and Taiwan coast,and eastern Fujian and southern Zhejiang coast.The mangroves in Beibuwan Gulf coast region took up 75.3% of the total mangrove germplasm resources in the country.At present,the percentage of the mangrove species applied for seedling propagation in China was estimated at 52.6%,most of which were of viviparous species.The six key steps in mangrove nursery operation included the selection of proper seedling propagation methods,the collection and storage of seeds or propagules,the ways of raising seedlings,the management of water and salinity,the control of diseases and pests,and the prevention of cold damage during winter.The structure,functions,and applications of the present five types of mangrove nurseries,including dry land nursery,mangrove tidal nursery,mudflat nursery,Jiwei pond nursery,and Spartina mudflat nursery,were also analyzed,which could provide guidance for the integrated management of mangrove ecological restoration engineering.林业科技支撑计划项目(2009BAD2B0605);国家海洋公益性行业科研专项经费项目(200905009)资

脑室下区胶质瘤病人生存状况的荟萃分析

目的通过Meta分析研究脑室下区(subventricular zone,SVZ)胶质瘤病人的生存状况。方法检索关于SVZ胶质瘤病人生存状况相关的文献,并按特定纳入标准与排除标准进行筛选。采用纽卡斯尔-渥太华量表对所选文献进行质量评价。使用随机效应模型或固定效应模型Meta分析比较SVZ胶质瘤和非SVZ胶质瘤病人中位生存期(overal survival,OS)和中位无进展生存期(progression free survival,PFS)的差异。结果本研究共纳入相关文献16篇。SVZ胶质瘤病人和非SVZ胶质瘤病人的中位OS分别为12.95个月和16.58个月,中位PFS分别为4.54个月和6.25个月。Meta分析结果显示:SVZ胶质瘤病人中位OS (OR=1.59,95%CI:1.35～1.87,P=0.036)及中位PFS (OR=1.44,95%CI:1.25～1.65,P <0.0001),均明显小于非SVZ胶质瘤。对选用多因素回归分析方法的文献进行亚组分析,亦发现SVZ胶质瘤病人中位OS明显小于非SVZ胶质瘤(OR=1.26,95%CI:1.12～1.42,P <0.0001)。结论 SVZ胶质瘤病人预后明显差于非SVZ胶质瘤病人。SVZ胶质瘤是影响胶质瘤病人预后的重要因素之一

Construction of SSH library from haemocyte of variously colored abalone challenged with bacteria and differential expression analysis of macrophage expressed protein

以雌性杂色鲍为对象,以大肠杆菌、副溶血弧菌、溶壁微球菌、表皮葡萄球菌、金黄色葡萄球菌的混悬液做为攻毒菌,利用抑制性差减杂交(SSH)技术构建细菌攻毒的杂色鲍血淋巴细胞SSHcDNA文库。随机挑取生长菌落110个克隆子,进行菌液PCR鉴定,计算文库重组率为98.18%,文库容量为1.37×106pfu。将重组子测序,经BLAST一致性搜索比对分析,有一重组片段含有穿孔素(Perforin)保守结构域,为巨噬细胞表达蛋白(MEP)类穿孔素部分cDNA序列,片段大小为1551bp,连续编码517个氨基酸残基,申请GenBank登录号为EU272049。经半定量PCR和荧光定量PCR差异显示分析,发现在细菌感染状态下MEP基因在血淋巴细胞中存在明显的上调表达现象。Abalones are considered to be the most precious delicacy from the sea, and become very important commercial seafood in aquaculture worldwide. Variously colored abalone (Haliotis diversicolor Reeve, 1846) has been widely cultured on the southeast coast for more than twenty years. However, abalone culture frequently suffers from bacterial infection and mass mortality of reared abalones causes serious economic losses. Unfortunately, knowledge of the defense mechanism in this animal is still lacking. In this study, using suppression subtractive hybridization (SSH) technology, a forward SSH li-brary was constructed from haemocytes of H. diversicolor, with the content of 1.37×106 pfu and the recombinant rate of98.18%. After the recombinant plasmids were sequenced, partial cDNA of macrophage expressed protein (MEP) was recognized based on BLAST searches in NCBI, with the size of 1 551 bp, and continuously encoding 517 amino acids. Semi-quantitative PCR and quantitative real-time PCR results showed that MEP cDNA was distinctly up-regulated in haemocytes of the bacterial-challenged group compared to the unchallenged group. The gene information obtained from this library will provide new insights into the immune mechanism of H. diversicolor and facilitate future study of target genes involved in the response to invading microorganisms.国家高技术研究发展计划项目(863计划)(编号:2007AA091406)资助~

The Effects of Benzo [a] Pyrene (BaP) Exposure on the CYP1A1 mRNA and AhR2 mRNA Expression of Red Seabream(Pagrus major)

通过水体暴露方式对海水养殖真鲷进行bAP持续染毒,利用实时定量PCr技术研究了真鲷细胞色素P450基因(CyP1A1)和芳香烃受体基因(AHr2)随bAP暴露剂量、时间的动力学变化。结果发现,0.1~1.5μg/l环境浓度的bAP能够显著性诱导CyP1A1基因和AHr2基因的表达,且AHr2 MrnA早于CyP1A1 MrnA被诱导表达;bAP持续暴露48 H,CyP1A1和AHr2基因的表达水平均随暴露时间的延长而显著升高,染毒72 H后又回复到本底水平,实验表明这两个基因的表达与bAP的暴露剂量和暴露时间之间具有显著性的剂量-效应和时间-效应关系。The gene expression patterns of CYP1A1 and Aryl hydrocarbon receptor(AhR2) of red seabream(Pagrus major) were both measured using real-time quantitative PCR(qPCR) when fish exposed to environmentally relevant concentration of BaP(0.1,0.5 and 1.0 μg/L,respectively).The results showed that CYP1A1 mRNA and AhR2 mRNA could be induced significantly,besides,the time of AhR2 mRNA induced ahead of the time of CYP1A mRNA induced.The two genes were induced markedly at the begining of BaP exposure,and then decreased to the basal levels after 72 h.The results demonstrate that BaP can regulate CYP1A1 and AhR2 transcript in a dose and time dependent manner.国家“863”计划重点资助项目(2007AA091406);国家自然科学基金资助项目(30770391

SARS-CoV N蛋白与人冠状病毒HCoV-OC43和HCoV-229E的交叉反应表位及特异表位的确定

为确定SARS-CoV N蛋白的特异抗原表位,对3种人冠状病毒SARS-CoV、HCoV-OC43和HCoV-229E N蛋白之间的交叉免疫反应进行了系统研究。构建了分别表达SARS-CoV、HCoV-OC43和HCoV-229E N蛋白的重组痘苗病毒,并制备了相应的小鼠免疫血清。用间接免疫荧光方法,检测了3种N蛋白的表达及其与3种冠状病毒免疫动物血清和SARS病人恢复期血清之间的反应。与此同时,用Western blot方法分析了原核表达的39个不同区段的SARS-CoV N蛋白与3种冠状病毒动物免疫血清和SARS病人恢复期血清之间的交叉反应性。免疫荧光检测结果表明,SARS-CoV、HCoV-OC43和HCoV-229E3种病毒的N蛋白在重组痘苗病毒感染的HeLa细胞中均可以特异表达;3种N蛋白之间存在明显交叉免疫反应。Western blot结果显示,SARS-CoV N蛋白的表位主要位于30~60aa、170~184aa、301~320aa和360~422aa;与HCoV-OC43的交叉反应表位主要位于30~60aa、90~120aa、204~214aa和320~360aa;与HCoV-229E的交叉反应表位主要位于30~60aa、150~160aa和301~360aa。含SARS-CoV N蛋白特异表位的重组肽N155b(60~214aa)和N185(30~214aa)只与SARS病人恢复期血清和灭活SARS-CoV免疫小鼠的血清反应,而不与灭活HCoV-OC43和HCoV-229E免疫的山羊血清产生交叉反应。上述结果为使用SARS-CoV N蛋白抗原进行特异诊断试剂的研究,提供了重要的实验依据

STUDY ON THE CONSTRUCTION AND IMMUNOLOGY CHARACTERISITCS of SIX-VA-LENT FUSION TOXIN of FOOD POISONING-BORNE BACTERIA

[目的]构建4种食源性致病菌融合毒素基因及重组表达载体,制备六联融合毒素的血清抗体。[方法]采用柔性lInkEr序列(g-g-g-g-S)对目的基因进行串联(HblA-VT1b-SEA-VT2b-bOnTAHC-SEb),构建重组表达质粒PET-22b(+)-f6并在E.COlI bl21中进行表达,将表达蛋白纯化后免疫豚鼠制备血清抗体,利用ElISA和琼脂扩散试验验证抗体的特异性与敏感性。[结果]成功构建了重组表达质粒PET-22b(+)-f6并在E.COlI bl21中成功表达,37℃表达蛋白主要以包涵体形式存在(表达量10.2%),基因序列全长3384bP,编码1127个氨基酸,蛋白分子量为127205,测序结果与设计序列一致性为100%。ElISA和琼脂扩散试验表明,融合毒素f6与4种食源性致病菌有良好的反应原性,与多种非目标菌不反应。[结论]成功构建了多联融合毒素基因的表达质粒及制备了抗血清,为利用融合毒素的方法检测食源性致病菌,进而建立食源性致病菌广谱、快速的检测方法奠定基础。[Objective] To construct toxin including fusion toxin gene from food poisoning bacteria and its recombinant expression vector,and then prepare serum antibody of Six-valent fusion toxin.[Methods] Six gene(HblA—VT1B—SEA— VT2B—BoNTaHc—SEB)fragments were connected by SOE-PCR via linker sequence encoding five amino acids(G-G-G-G-S),the recombination expression plasmid pET-22b(+)-F6 was constructed and expressed in E.coli BL21.The expression proteinum was purified,and the blood serum was prepared by the immune cobaya,The specificity and sensitivity of antibody were verified by the ELISA and agar diffusion reaction.[Results] The fusion gene F6(HblA—VT1B—SEA—VT2B—BoNTaHc—SEB)and re-combinant plasmid pET-22b(+)-F6 was successfully constructed.The most of the 37℃ expression proteinum showed to be cyt-orrhyctes(expreesion amounted to 10.2%).The sequence encoding the mature fusion protein of the F6 toxin gene was 3384bp,encoding 1127 amino acids.The molecular weight of recombinant fusion toxin protein was 127.205 ku.The result of sequencing was consistent with predicted gene sequences.The results of ELISA and agar diffusion reaction demonstrated that fusion gene F6 and other four kinds of food poisoning-born bacteria had favorable reactionogenicity and did not response to many other non-tar-get bacteria.[Conclusion] The expression plasmid of multi-valent fusion toxin was successful constructed and antiserum was prepared,which laid the foundation for establishing broad spectrum and rapid detection method for food poisoning bacteria by utilization of fusion toxin detection methods.国家自然基金资助项目(30671762);厦门市科技计划项目(3502Z20055009