Cloning and Expression of the Leukotoxin BSBSE Gene from Fusobacterium necrophorum

以牛源坏死梭杆菌FNn株为试材,根据GenBank上已发表的坏死梭杆菌AF312861标准菌株的lktA序列设计1对引物,利用PCR技术扩增出1 131 bp的坏死梭杆菌白细胞毒素BSBSE基因。将PCR产物插入pGEM-T Easy vector中,经双酶切鉴定正确后进行序列测定。分析表明该BSBSE序列与GenBank上已发表的坏死梭杆菌AF312861标准菌株的lktA序列的核苷酸同源性为99%,推导出的氨基酸序列同源性为98%。为研究BSBSE的免疫原性,构建了原核表达载体pMAL-p2X-BSBSE,用IPTG诱导在大肠杆菌中表达。结果表明,BSBSE基因在大肠杆菌中进行了高效特异性融合表达,融合蛋白分子量约为84.5×103,其中41.5×103为BS-BSE基因表达的蛋白质,43.0×103为MBP融合标签,Western-blotting检测表明该表达产物有免疫原性。According to the sequence of announced lktA gene in Fusobacterium necrophorum,a pair of primers were designed.The BSBSE gene was amplified by PCR.The product was cloned into pGEM-T Easy vector.When nucleotide sequence and deduced amino acid sequence were compared with homologous sequence of the FN AF312861 lktA of GenBank,the homologue of the mucleotide sequence is 99% and the homologue of the amino acid sequence is 98%.The BSBSE fragment was inserted into expression vector pMAL-p2X and the plasmid pMAL-p2X-BSBSE were expressed in E.coli BL21 by IPTG induction.The SDS-PAGE analysis indicated the weight of the fusion protein was about 84.5.0×103,which included the 41.5×103 protein expressed from BSBSE gene and 43.0×103 fusion MBP tag.The recombinant BSBSE-pMAL-p2X production has Immunogenicity with western-blotting.The cloning and expression of the BSBSE gene established the foundation of further research on the function and application of the BSBSE gene.“十五”国家科技攻关子课题(2002BA518A04);; 中国农业科学院特产研究所科研基金项目(Tcs2005-03

Cloning and Expression of the Leukotoxin Gene SH from Fusobacterium necrophorum

坏死梭杆菌白细胞毒素是坏死杆菌病的主要致病因子,白细胞毒素基因(lkT)是其编码基因。以分离到的国内牛源坏死梭杆菌fn(A)菌株f4基因组dnA为模板,应用PCr方法扩增白细胞毒素基因SH片段,克隆至PMd18-T载体上,以bAMHⅠ和HIndⅢ酶切的目的片段SH与相应酶切的PET32A载体连接构建PET32A-SH重组表达质粒,经转化E COlI bl21(dE3)后用IPTg进行蛋白诱导,SdS-PAgE检测重组蛋白表达情况。结果表明:扩增基因序列大小为1800bP,SdS-PAgE检测重组蛋白有效表达,表达得到大小为80.2kdA的目的蛋白,采用镍柱亲和层析方法纯化SH重组蛋白,获得了纯度达95%的重组蛋白;经WEST-Ern-blOT证实,该蛋白对抗坏死杆菌阳性血清具有反应活性。The leukotoxin of Fusobacterium necrophorum(FN) is considered to be one of the main virulence factors.The lkt gene encodes for FN.In this study,the SH fragment of lkt gene was amplified by PCR using the F4 genome as the template,which was isolated from the Chinese Fusobacterium necrophorum strain.The fragment was then cloned to the pMD18-T vector for sequencing.Thereafter,the SH fragment was subcloned into the multiple cloning sites of the pET32 to construct pET32a-SH recombinant plasmid,which was then trans-formed into E.coli BL21(DE3) with IPTG induction for expression.SDS-PAGE was used to analyze the recombinant protein.The results showed that the SH fragment of about 1800 bp was amplified and was about 80.2 kDa.The fusion protein was purified by Ni-NTA affinity chromatography under denature conditions,and their purity was above 95%.Western-blot analysis indicated the SH fragment had anti-genicity against Fusobacterium necrophorum.“十五”国家科技攻关子课题(2002BA518A04);吉林省科技发展计划项目(20070570);吉林市科技发展计划项目(200805

The effects of ocean acidification on marine organisms and ecosystem

海洋酸化是CO2排放引起的另一重大环境问题.工业革命以来,海洋吸收了人类排放CO2总量的三分之一.目前,海洋每年吸收的量约为人类排放量的四分之一(即约每小时吸收100万吨以上的CO2),对缓解全球变暖起着重要的作用.然而,随着海洋吸收CO2量的增加,表层海水的碱性下降,引起海洋酸化.海洋酸化会引起海洋系统内一系列化学变化,从而影响到大多数海洋生物的生理、生长、繁殖、代谢与生存,可能最终导致海洋生态系统发生不可逆转的变化,影响海洋生态系统的平衡及对人类的服务功能.地球历史上曾多次发生过海洋酸化事件,伴随着生物种类的灭绝,其内在联系虽然不甚明确,却也可能暗示未来海洋酸化可能对海洋生态系统产生重大的影响.Ocean acidification is known as another global change problem caused by increasing atmospheric CO2.Since the industrial revolution, the oceans have absorbed more than one third of the anthropogenic CO2 released to the atmosphere, currently, at a rate of over 1 million tons per hour, totaling to about one quarter of all anthropogenic CO2 emissions annually.Uptake of CO2 by the ocean has played an important role in stabilizing climate by mitigating global warming.However, rising ocean carbon levels caused by the uptake of anthropogenic CO2 (acidic gas) leads to increased ocean acidity (reduced pH) and related changes in ocean carbonate chemistry, or "ocean acidification".Recent research has shown that ocean acidification affects the physiology, growth, survival, and reproduction of many, if not most marine organisms.Ultimately, future ocean acidification may lead to significant changes in many marine ecosystems, with consequential impact on ecosystem services to societies.Several ocean acidification events are known to have occurred during Earth’s history, each coinciding with high rates of species’ extinctions.Although the mechanisms involved in past massive species extinction associated with ocean acidification events, they certainly hint potential disastrous impacts on ecosystem functions in short future.中国科学院“百人计划”(2006-067); 国家自然科学基金(40872168)资

ECFA之后的两岸政治关系走向

在两岸政治互信和良性互动逐渐增强、两岸全面交流局面形成、两岸顺利签署ECFA的大背景下,两岸关系和平发展如何向深度和广度推进,特别是如何发展两岸政治关系?成为两岸有识之士普遍关注的话题。两岸著名专家学者发表看法

一种超小硅基多波长路由器的仿真研究

文章设计了一种超小的硅基多波长路由器,它可实现光通信窗口多波长的路由功能。首先用平面波展（PWG）方法分析了这种光路由的理论及其结构特性,然后用时域有限差分（FDTD）方法数值模拟了该类器件的导光波特性及光场能量传播特性。所设计的硅基多波长路由器具有透射效率高和整体尺寸超小（总长度在20μm左右）的特点,能同时实现1.31、1.55和1.65μm光波长的路由功能

PCR-SSP 快速HLA-DR 基因分型法#br# 的建立及临床初步应用

摘　要　采用序列特异性引物聚合酶链反应(PC R-SS P)方法, 对22 份国际标准细胞株DNA 及23 例临床 血标本DNA 进行H LA-DR 基因分型, 扩增条件为94 ℃, 30 s , 58 ℃, 30 s 共30 个循环, 对各个标准DNA 的分 型结果显示, 符合率为100 %, 无假阳性及假阴性,提示本方法快速、准确, 适合于临床应用

Cloning and Sequencing of Pili Gene of D.nodosus Serotype A in Footrot

以腐蹄病A型节瘤拟杆菌染色体DNA为模板,根据设计的上、下游引物进行PCR反应,扩增出了大小约0.78kb的基因片段,用T_4DNA连接酶将PCR产物与pGEM-T-Easy载体连接,构建了重组质粒。并以EcoRV和SalI双酶切重组质粒鉴定目的基因的插入方向。经序列测定,克隆的目的基因片段为Pili基因。The pili gene that dominates the main protective antigen was amplified and cloned from D. nodosus serotype A by PCR. A recombinant plasmid,designated TE- Pili,was constructed by inserting the Pili gene intoT - Easy which was digested with EcoRV and Sal I. The result of sequencing showed that the fragment was exactly Pili gene of D. nodosus serotype A.国家科技部攻关计划“奶牛主要疫病防治关键技术研究与产业化开发”项目(2002BA518A04

Establishment of a PCR Technique to Detection of I,II Groups of D.nodosus in Footrot in Ruminants

根据节瘤拟杆菌特有保守序列设计引物,利用聚合酶链式反应(PCR)建立了一种鉴别检测该菌的方法。对可能影响PCR检测结果的一系列因素进行了较详细的研究,选择了适宜PCR检测的快捷处理病样获得反应模板的方法,对PCR反应条件进行了优化,使对已知菌株培养物检测灵敏性最高可到30个菌/30μL反应体系;用广泛的相关菌株和菌群验证引物的特异性,证明设计的引物特异性强。将PCR方法用于临床病样的初步检测,部分病样PCR检测阳性。为用PCR方法检测节瘤拟杆菌引发的牛、羊、鹿腐蹄病奠定了基础。Detection assay by PCR(Polymerase chain reaction) was constructed to D.nodosus,that can discriminate two group of Dichelobacter nodosus:(1)Three primers were designed,based on the conserved region of aligned fimA sequences of groups I,II of D.nodosus,of which forward primers were identical to group I,II,and reverse primers was specific to each group I and group II respectively;(2)The optimal PCR conditions were determined,in 30μl reaction volume the primers detected at least 30 cells of D.nodosus in crude lysates;(3)A serial of PCR assay verified the primers was specific to D.nodosus but not to the other bacterial and cells.The developed PCR technique were evaluated with directly lysate of samples from lesions footrot,the results found a few sample were positive in PCR test.From the results,which is suggested the developed PCR technique is adapted to identify D.nodosus and discriminate its serogroups,It will become a practical method to diagnosis of footrot in ruminants.中国科技部奶业专项“奶牛蹄病高效疫苗的研究与产业化开发”(2002BA518A04

广州市至2020年城市客运交通发展情景分析

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重大工程地质灾害的预测理论及数值分析方法研究年度报告

基本完成了SAP84-NOLM的PC版本并进行了测试,可以实现在PC上计算大约30万自由度的地质体结构。其中包括带有裂隙的地质体的初始网格生成和裂隙扩展时的网格自适应;2D/3D渗流场的计算;用弧长法来计算材料的软化,以考虑水对地质体本构的影响以及大规模并行计算的初步实现。小尺寸滑坡物理测试平台已经完成并进行了实验,大尺寸滑坡物理测试平台正在施工建设中