脂多糖对大黄鱼原代头肾巨噬细胞的激活作用

为了揭示硬骨鱼C型凝集素受体 (C-type lectin receptor,CTLR)的生物学功能,实验以从大黄鱼转录组数据库中筛选出的一个CTLR基因—C型凝集素结构域家族4成员E基因 (Clec4e)为研究对象,研究其分子特征、表达分布和凝集特性。结果显示,LcClec4e cDNA全长1 546 bp,开放阅读框 (ORF)771 bp,编码254个氨基酸。LcClec4e的N端有一个跨膜区,无信号肽,C端含有一个糖识别结构域 (CRD),其中含有糖结合位点EPN和WFD以及6个可形成二硫键的保守半胱氨酸。系统发育分析表明,LcClec4e与多种鲈形目鱼类Clec4e具有较近的亲缘关系。荧光定量PCR结果显示,LcClec4e在所检测的10种组织中呈组成型分布,且在肝脏中表达量最高;LcClec4e在来源于大黄鱼头肾组织的原代巨噬细胞、淋巴细胞和粒细胞中均有表达,且在巨噬细胞中表达量最高;经灭活溶藻弧菌刺激后,LcClec4e在3种免疫细胞中的表达均极显著上调。原核表达的重组LcClec4e胞外段 (recombinant LcClec4e-extracellular domain,rLcClec4e-ex)具有Ca²⁺依赖性的凝集活性,可凝集小鼠、家兔的红细胞,以及嗜水气单胞菌、变形假单胞菌、溶藻弧菌和坎氏弧菌等4种水产常见的革兰氏阴性菌。D-葡萄糖、D-果糖、D-甘露糖、D-麦芽糖、α-乳糖和脂多糖均可抑制rLcClec4e-ex对大黄鱼重要病原菌变形假单胞菌的凝集作用,说明LcClec4e可能与变形假单胞菌表面的糖类物质结合。上述结果提示,LcClec4e可能作为一种模式识别受体,通过结合病原菌表面的糖类病原体相关分子模式来识别病原,参与大黄鱼抗细菌感染的免疫防御。

     

福建鳗鲡产业发展形势分析

为建立美洲鳗鲡腺瘤病毒 (AEAdoV)的检测方法,根据AEAdoV福建株 (AEAdoV-FJ)的superfamily 3 helicases (S3H)序列,设计引物,建立了AEAdoV的普通PCR和qPCR检测方法;进一步评价检测方法的灵敏性、特异性及重复性,利用2种方法对美洲鳗鲡“出血性烂鳃”病料进行了检测,并对美洲鳗鲡体内不同组织的病毒含量进行分析。结果显示,普通PCR扩增的目的片段长度约300 bp,利用其构建的qPCR质粒标准品,其拷贝数与qPCR阈值循环数(C_t)线性关系良好,线性范围广,标准曲线相关系数(R²)达到0.999,扩增效率为105.067%。建立的普通PCR法和qPCR的最低检测AEAdoV拷贝数分别为100个和10个。2种方法均可特异性检测AEAdoV,而对蛙虹彩病毒 (RGV)、鳗鲡疱疹病毒 (AngHV)、鲤疱疹病毒 (KHV)、对虾白斑综合征病毒 (WSSV)、日本鳗鲡内皮细胞坏死病毒(JEAdoV)和花鳗鲡腺瘤病毒(MEAdoV)均无扩增反应。qPCR法的组内和组间变异系数均小于2%,表明其重复性良好。临床应用结果显示,35份美洲鳗鲡“出血性烂鳃”病料,采用普通PCR法的AEAdoV检出率为82.8%,而qPCR法的AEAdoV检出率为97%。对美洲鳗鲡不同组织的病毒含量分析结果显示,心脏、肝脏、鳃、鳍的AEAdoV相对含量较高,而黏液、皮肤和脾脏的病毒含量相对较低。研究表明,建立的AEAdoV的灵敏度高、特异性强的普通PCR和qPCR检测方法,证实AEAdoV与美洲鳗鲡“出血性烂鳃”病密切相关,且在感染鳗鲡主要组织中都存在。实验结果对于研究AEAdoV的致病性,开展其流行情况和病原学情况分析具有重要意义。

     

永靖县发展休闲渔业对策探讨

论文对甘肃省永靖县休闲渔业的发展优势与现状进行了系统调查,指出了休闲渔业在永靖县传统渔业向现代渔业转型发展过程中的重要作用。分析了休闲渔业存在的问题:休闲渔业布局不合理,缺乏规划;宣传不到位,基础设施较薄弱;从业人员素质不高,创新意识不强。并在此基础上提出了发展对策:一是确立休闲渔业的主导产业地位,提高群众和从业者素质;二是必须坚持生态发展休闲渔业理念;三是建设两大休闲渔业基地;四是实施休闲渔业场所的监管和评级制度。     

Analysis on Target Genes with Mutation in microRNA of Blue-shelled Chicken and White Leghorn

The study was conducted to explore the potential different characters between Blue-shelled chicken and White leghorn.Global genome microRNA was combined the identified microRNA with complementary lab-predicted microRNA.Then the two breed chicken's SNP data got by GGRS were mapped to the microRNA and focused on SNP that deliberately located in mature-microRNA.Bioinformatics method was adopted for target prediction on microRNA which had SNPs.By further gene enrichment analysis,the study found these genes enriched in 22 GO terms,10 KEGG pathways,and 3 IPA important networks.And they enriched in traits which associated with growth,such as mTOR signaling pathways,Wnt signaling pathways,growth hormone receptor networks and insulin-like growth factor Ⅰ receptor networks.And they also enriched in some laying traits,such as oocyte meiotic signaling pathways and progesterone mature oocytes signaling pathways.The methods and the results might provide references for further studies.     

Effect of AIR on Inflammatory Reaction Infected by Brucella

In order to investigate the effect of AIR on inflammatory reaction infected by Brucellamelitensis (16M), the AIR domain of Tecpr1 gene of murine macrophages RAW264.7 were knocked down (I-A), overexpressed (O-A) and reversed (OA-IA). Using the chlorine fluorescein (DCFH-DA) as a probe, we detected the variation of ROS production and mitochondria distribution by confocal laser scanning microscopy. We observed the expression changes of NLRP3, ASC and Caspase-1 by qRT-PCR and the expression changes of IL-18,IL-1β and Caspase-1 in host cells by ELISA. The results showed that 16M could stimulate RAW264.7 cells to produce ROS by time-dependent pathway, and I-A group and O-A group showed more abnormal accumulation of mitochondrial. The results of qRT-PCR and ELISA suggested that it had effect on the expression levels of NLRP3, ASC,Caspase-1 and IL-18, IL-1β and Caspase-1 in cells of different groups. Those results indicated that with AIR gene deletion, the release amount of ROS changed, mitochondrial clustered abnormally, and AIR was closely related to the activation of inflammasomes and induction of inflammatory reactions.     

Effect of Luteolin on Blood Indexes,Liver and Kidney in Mice with Acute Mercury Poisoning

To study the effect of luteolin on blood indexes,liver and kidney in mice with acute mercury poisoning,28 mice were randomly divided into four groups:Control group (intraperitoneal injection 0.9% saline),luteolin group (lavage 100 mg/kg luteolin),mercuric chloride group (intraperitoneal injection 4 mg/kg mercury chloride) and mercury chloride+luteolin group (intraperitoneal injection 4 mg/kg mercuric chloride,lavage 100 mg/kg luteolin).The activities of ALT in serum,AST,CREA and BUN contents,blood WBC,RBC,HGB content and GSH and MDA contents of liver tissue were detected.Morphological changes of liver and kidney tissues were observed.The results showed that compared with the control group,the activities of ALT and AST of mercuric chloride group were extremely significantly increased (P < 0.01),serum CREA,BUN,blood WBC and liver tissue MDA contents were significantly increased (P < 0.05) while blood RBC,HGB and liver tissue GSH contents were significantly decreased (P < 0.05).Liver,kidney pathological changes were obviously.Compared with mercuric chloride group,the activities of ALT,AST in serum,CREA,BUN,blood WBC and liver tissue MDA contents of mercuric chloride+luteolin group were significantly decreased (P < 0.05),while blood RBC,HGB and liver tissue GSH contents were significantly elevated (P < 0.05).Liver,kidney pathological changes were attenuated obviously.The poisoning were characterized by inflammation and the occurrence of anemia when acute mercury poisoning occurred,liver and kidney showed different degrees of injury in mice.Luteolin could reduce the toxic effects of acute mercury poisoning on blood,liver and kidney.     

用于构建双价禽流感DNA疫苗的双目的基因表达质粒的构建及鉴定

为探索构建禽流感双价或多价DNA疫苗的可行性,本研究以本实验室前期研制的禽流感DNA疫苗p CAGGopti HA5为基础[其中含有禽流感病毒(AIV)A/Goose/Guang Dong/1/96(H5N1)的HA基因],将增强型绿色荧光蛋白(eGFP)报告基因作为第二个目的基因,利用双启动子启动、内部核糖体进入位点(IRES)介导以及通过蛋白Linker编码序列连接融合表达两个目的基因的形式,构建了双启动子重组表达质粒pH5-eGFP、IRES介导的重组表达质粒pHIE以及融合表达重组质粒pH6E。并采用脂质体介导将这3种重组质粒分别转染293T细胞,通过间接免疫荧光试验和western blot方法检测HA和eGFP这两种目的蛋白的瞬时表达情况。结果显示,这3种重组质粒均能够正确表达AIV的HA蛋白和eGFP。其中pH5-eGFP表达出两个独立的目的蛋白,而且互相不影响各自的表达和细胞定位;pH6E则以融合形式表达两种目的蛋白,由于HA蛋白具有膜定位信号,所以融合蛋白主要定位于细胞膜上。本研究结果表明,双启动子表达质粒和双目的蛋白融合表达质粒适合用于构建AIV以及其他相关病原的双价DNA疫苗。     

关于生物类基础化学教学中应用“问题学习法”的探讨

本文通过课前、课堂、课后三个部分的问题设置,紧扣教学大纲,紧密联系中学化学和后续课程中相关知识,体现理论联系实际、学科前沿。引领学生主动学习,积极思考,为后续课程学习起到积极的、有效的铺垫作用。