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1.
为构建安全高效表达海洋源金属硫蛋白的益生菌重组菌株,以海洋源褶牡蛎内脏团为模板,PCR克隆得到褶牡蛎金属硫蛋白基因(Op-MT),大小为324 bp,经pMD19-T载体亚克隆到融合表达载体pHT43-SUMO中,然后通过电转化法将重组质粒转入八种蛋白酶缺陷的宿主枯草芽孢杆菌(Bacillus subtilis)WB800N中。SDS-PAGE电泳显示诱导表达的融合蛋白SUMO-Op-MT分子量约为23 kD,与预期一致,说明可溶性融合蛋白SUMO-Op-MT在宿主菌中得到了表达,且通过Western blotting进一步得到验证。同时,HPLC检测重组枯草芽孢杆菌发酵液,结果也表明重组枯草芽孢杆菌成功表达了可溶性目的蛋白Op-MT。通过对重组工程菌中MT活性检测发现,其对重金属镉耐受力增强,说明重组工程菌成功表达出有活性的金属硫蛋白。这为以后开发天然、高效海洋源重金属解毒剂提供理论基础。  相似文献   

2.
对斜带石斑鱼(Epinephelus coioides)的干扰素调节因子1(Interferon regulatory factor 1,IRF-1)基因进行了克隆、测序及原核表达.以PBS做对照,利用polvI:C刺激斜带石斑鱼,然后取其肝脏、头肾、脾脏提取总RNA,随机引物反转录获得cDNA.根据相近物种IRF-1的保守序列设计引物,通过PCR得到了保守片段,通过RACE-PCR获得了斜带石斑鱼IRF-1基因的全长cDNA.基因全长为1 730 hp,完整开放阅读框(ORF)906 bp,编码302个氨基酸,5'非编码区153 bp,3'非编码区671 bp.将斜带石斑鱼ORF与其他物种进行比对发现,与海鳜(Siniperca chuatsi)同源性最高,为85%.将根据斜带石斑鱼IRF-1 ORF推测的氨基酸序列与其他物种进行比对,发现斜带石斑鱼与海鳜同源性为90%,与金头鲷(Sparus aurat)为88%,与乌鳢(Channa argus)为86%,与大菱鲆(Scophthalmus maximus)为82%.利用ORF序列,构建原核重组表达质粒.重组质粒经PCR、酶切鉴定及测序验证后转化大肠杆菌BL21(DE3),经IPTG诱导,重组蛋白成功表达,表达蛋白的分子量约55 kD.诱导温度为30℃时,重组蛋白以可溶性蛋白和包涵体2种形式存在.  相似文献   

3.
根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到自杀性DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)自杀性DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但自杀性DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。  相似文献   

4.
金属硫蛋白(Metallothionenin,MT)是一类普遍存在于生物体内、分子量低、半胱氨酸含量丰富、能够与二价重金属离子结合的蛋白质.MT在清除自由基、解除重金属毒性、参与体内微量元素代谢、防止细胞癌变等方面具有重要作用.本研究通过构建大珠母贝外套膜全长cDNA文库,首次克隆得到大珠母贝金属硫蛋白(PmMT)全长cDNA序列.该序列全长599bp,5'UTR (Untranslated Region)为75bp,3'UTR为296 bp,开放阅读框(Open Reading Frame,ORF)为228bp,编码75个氨基酸.编码的氨基酸中半胱氨酸含量丰富,达到29.3%;赖氨酸和甘氨酸含量也较高,均为9.3%;不含苯丙氨酸、组氨酸、色氨酸、酪氨酸等芳香族氨基酸;含有软体动物等无脊椎动物金属硫蛋白的特征序列.序列特征分析表明,该序列具备金属硫蛋白的典型特征,是金属硫蛋白家族成员.  相似文献   

5.
根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到"自杀性"DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)"自杀性"DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但"自杀性"DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。  相似文献   

6.
Ⅱ型鲤疱疹病毒ORF121蛋白的多克隆抗体制备及鉴定   总被引:1,自引:0,他引:1       下载免费PDF全文
余琳  吕利群  王浩 《水产学报》2019,43(6):1463-1471
针对CyHV-2病毒ORF121基因(GenBank:AFJ20543.1)进行原核表达系统的构建,将纯化重组蛋白作为抗原来免疫BALB/c小鼠获得多克隆抗体,应用该抗体开展CyHV-2病毒诊断及其感染机制研究。以CyHV-2病毒感染细胞上清液为扩增模板,扩增ORF121基因构建至pGEX-4T原核表达载体,经异丙基硫代半乳糖苷(IPTG)诱导表达rORF121重组蛋白,利用尿素纯化后免疫6周龄BALB/c小鼠制备多克隆抗体。结果显示,CyHV-2病毒ORF121基因可在原核表达系统中高效表达目的重组蛋白rORF121,经SDS-PAGE分析大小约为60 ku,主要以不可溶的包涵体存在。利用尿素溶解rORF121蛋白免疫BALB/c小鼠获得抗ORF121蛋白的多克隆抗体,Western Blot实验显示,该抗体可特异性识别CyHV-2病毒感染RyuF-2细胞样品。研究表明,利用CyHV-2感染RyuF-2细胞后,本研究制备的抗ORF121蛋白的多克隆抗体能够通过间接免疫荧光实验特异性识别CyHV-2病毒感染的细胞样品。本研究制备的抗ORF121蛋白的多克隆抗体,能够为CyHV-2病毒诊断技术的构建以及深入开展CyHV-2病毒感染机制提供良好的技术基础。  相似文献   

7.
根据已登录的中华绒螯蟹一种Δ6去饱和酶基因(Accession Number:JX946434)及菁夜蛾去饱和酶基因(Accession Number:KJ622055.1)的保守区设计引物,通过逆转录PCR以及RACE技术克隆了中华绒螯蟹另一种Δ6去饱和酶基因FAD6-b的全长序列。经序列分析,中华绒螯蟹FAD6-b cDNA全长为2 310 bp,其中开放阅读框(ORF)为1 326 bp,共编码442个氨基酸(Accession Number:KP876058),理论分子量为50.86 ku,理论等电点为8.47,FAD6-b基因的氨基酸序列与已公布的一条中华绒螯蟹Δ6去饱和酶基因的一致性为76%。原核表达载体构建及其表达实验表明,中华绒螯蟹FAD6-b基因成功重组到原核表达载体pCold-TF DNA中,重组质粒pCold TF-fad6b在大肠杆菌BL21(DE3)pLysS中进行表达,经SDS-PAGE电泳分析表明,IPTG诱导后的重组菌出现了单一的蛋白条带,且大小与预期(105.86 ku)一致,可溶性分析显示目的蛋白主要存在于蛋白上清液中。对重组蛋白进行纯化,蛋白纯化液经电泳检测后显示出特异性单一条带,进一步证明了pCold TF-fad6b的成功构建和表达。目的蛋白经Western-blotting检测,结果表明获得的重组蛋白与6×His抗体进行了特异性结合,显示出良好的免疫学活性。  相似文献   

8.
金属硫蛋白(Metallothionein,MT)是一类普遍存在于生物体内、分子量低、半胱氨酸含量丰富、能够与二价重金属离子结合的蛋白质。MT在清除自由基、解除重金属毒性、参与体内微量元素代谢、防止细胞癌变等方面具有重要作用。本研究通过构建大珠母贝外套膜全长cDNA文库,首次克隆得到大珠母贝金属硫蛋白(PmMT)全长cDNA序列。该序列全长599bp,5’UTR(Untranslated Region)为75bp,3’UTR为296bp,开放阅读框(Open Reading Frame,ORF)为228bp,编码75个氨基酸。编码的氨基酸中半胱氨酸含量丰富,达到29.3%;赖氨酸和甘氨酸含量也较高,均为9.3%;不含苯丙氨酸、组氨酸、色氨酸、酪氨酸等芳香族氨基酸;含有软体动物等无脊椎动物金属硫蛋白的特征序列。序列特征分析表明,该序列具备金属硫蛋白的典型特征,是金属硫蛋白家族成员。  相似文献   

9.
根据已克隆的半滑舌鳎(Cynoglossus semilaevis)生长激素(GH)基因和生长激素受体Ⅰ(GHR-Ⅰ)基因的cDNA序列信息设计引物,用于扩增半滑舌鳎GH、GHR-Ⅰ基因序列。随后,分别利用表达载体pET-32a和pGEX-6P-1成功构建了包含半滑舌鳎GH、GHR-Ⅰ基因ORF全序列及不同ORF片段的6个重组质粒,并将获得的重组质粒在表达宿主菌大肠杆菌(Escherichia coli)BL21(DE3)中,用IPTG诱导表达。SDS-PAGE电泳结果显示:重组GH的pET-32a-CSGH质粒在40 kD处有特异性的蛋白条带,重组GHR-Ⅰ胞内区的pGEX-6P-1-CSGHR1-2质粒在70 kD处发现诱导的蛋白条带,其表达量分别占细胞蛋白总量的37.6%和20.2%;而构建的pGEX-6P-1-CSGH重组质粒和含有GHR-Ⅰ基因跨膜区的重组质粒(pET-32a-CSGHR1-1、pET-32a-CSGHR1-2和pGEX-6P-1-CSGHR1-1)均未获得表达产物。由此可见,半滑舌鳎GHR-Ⅰ基因的跨膜区可能影响其在大肠杆菌中的表达。研究结果为实现半滑舌鳎GH、GHR-Ⅰ制剂的开发及应用于养殖生产实践奠定基础。  相似文献   

10.
锦鲤疱疹病毒ORF72基因克隆及结构功能分析   总被引:1,自引:0,他引:1  
于辽宁丹东采集感染锦鲤疱疹病毒的活鲤Cyprinus carpio样品,经DNA提取、PCR扩增、重组质粒构建步骤成功克隆锦鲤疱疹病毒ORF72基因,并利用生物信息学软件分析ORF72蛋白结构与功能,构建系统进化树。结果显示:ORF72基因开放阅读框共1113bp,编码蛋白由370个氨基酸组成,理论相对分子质量为40.5KD;ORF72蛋白主要由α螺旋、β折叠、无规则卷曲3种二级结构组成;N端35个氨基酸序列为信号肽序列。跨膜结构分析表明:ORF72蛋白无跨膜区,保守结构域预测表明ORF72含有一个保守结构域,抗原表位分析表明,抗原性较好。结构预测显示:ORF72氨基酸序列存在2个潜在的N-糖基化位点、1个潜在的O-糖基化位点、19个潜在的磷酸化位点。系统进化树分析发现,其与锦鲤疱疹病毒美国株、以色列株属同一分支。  相似文献   

11.
文蛤金属硫蛋白基因的克隆与分析   总被引:3,自引:0,他引:3  
金属硫蛋白是一种普遍存在于生物体内的低分子量、半胱氨酸含量丰富、易于被外界刺激诱导的金属结合蛋白。采用表达序列标签(EST)法,首次获得了文蛤金属硫蛋白(Mm—MT)的全长eDNA序列,序列全长657bp,开放阅读框长度为231bp,可以编码76个氨基酸。编码的氨基酸中半胱氨酸含量丰富,达27.6%,含有软体动物等无脊椎动物金属硫蛋白的特征序列。序列特征分析表明,该序列具备金属硫蛋白的典型特征,是金属硫蛋白家族成员。  相似文献   

12.
Considerable time and money are expended in the pursuit of catching fish with hooks (e.g., handlining, angling, longlining, trolling, drumlining) across the recreational, commercial and subsistence fishing sectors. The fish and other aquatic organisms (e.g., squid) that are captured are not a random sample of the population because external (e.g., turbidity) and underlying internal variables (e.g., morphology) contribute to variation in vulnerability to hooks. Vulnerability is the probability of capture for any given fish in a given location at a given time and mechanistically explains the population‐level catchability coefficient, which is a fundamental and usually time‐varying (i.e., dynamic) variable in fisheries science and stock assessment. The mechanistic drivers of individual vulnerability to capture are thus of interest to fishers by affecting catch rates, but are also of considerable importance to fisheries managers whenever hook‐and‐line‐generated data contribute to stock assessments. In this paper, individual vulnerability to hooks is conceptualized as a dynamic state, in which individual fish switch between vulnerable and invulnerable states as a function of three interdependent key processes: an individual fish's internal state, its encounter with the gear, and the characteristics of the encountered gear. We develop a new conceptual framework of “vulnerability,” summarize the major drivers of fish vulnerability, and conclude that fish vulnerability involves complex processes. To understand vulnerability, a shift to interdisciplinary research and the integration of ecophysiology, fish ecology, fisheries ecology and human movement ecology, facilitated by new technological developments, is required.  相似文献   

13.
Metallothioneins (MTs), considered as metal biomarkers by many authors, are very conserved proteins that present great interests in phylogenetical studies. This paper reports the purification of the hepatic MT from the roach (Rutilus rutilus). After extensive reverse-phase HPLC, the sequence of the MT was established by a combination of techniques including automated Edman degradation, mass spectrometry and enzymatic treatment. Sequence comparison with other fishes or mammalian MTs showed considerable identities and a high conservation in cysteine residues positions. MTs of roach and goldfish that both belong to the same family (Cyprinidae) share 97% identity. Ninety five% identity were found between roach and stone loach (Cypriniforms, Cobitidae) MTs. Only 77% identity were found with pleuronectiforms or salmoniforms. This sequence brings a new element in the study of the phylogenetical relationships among teleost species.  相似文献   

14.
微量元素对水华发生、发展的影响   总被引:7,自引:0,他引:7  
微量元素作为藻类营养因子的一部分,对藻类生长具有重要作用,而藻类(特别是微囊藻)的大量增长则会造成"水华"现象。本文综述了各种微量元素对水华藻类生长的影响及其影响机理,进而得出结论:微量元素对水华藻类的生长影响趋势为低浓度促进生长,高浓度抑制生长,而藻类对微量元素有一定的富集作用;在微量元素中,铁元素对水华的发生、发展至为重要,在某种情况下有可能成为水华发生、发展的限制因子;其他元素(如稀土元素和重金属元素等)在水华发生、发展中则起辅助作用。  相似文献   

15.
Understanding drivers of freshwater fish assemblages is critically important for biodiversity conservation strategies, especially in rapidly developing countries, which often have environmental protections lagging behind economic development. The influences of natural and human factors in structuring fish assemblages and their relative contributions are likely to change given the increasing magnitude of human activities. To discriminate natural and human drivers of fish diversity and assemblage patterns in developing countries with rapid socio‐economic development, a dataset of 908 freshwater fish species and 13 metrics including three categories of both natural (i.e., biogeographic) and human drivers (i.e., economic growth, inland fisheries) in China were analysed with machine learning algorithms (i.e., self‐organizing map, random forest). Here, we found that biogeographic drivers explained 21.8% of the observed fish assemblage patterns in China and remained stronger predictors when compared to human drivers (i.e., 15.6%, respectively). Freshwater fish species richness was positively correlated to rainfall, air temperature, surface water area and inland fisheries production but negatively correlated with urbanization. In addition, the strong structuring effects of climatic variables on Chinese fish richness patterns suggested that the fish assemblages could be particularly vulnerable to climate change. Our results showed that natural biogeographic factors still dominate in driving freshwater fish assemblage patterns despite increased human disturbances on aquatic ecosystems in a rapidly developing country. These findings consequently suggested that we should consider both natural (e.g., climate) and human (e.g., urbanization, inland fisheries) factors when establishing aquatic conservation strategies and priorities for developing countries that are experiencing rapid socio‐economic changes.  相似文献   

16.
Marine protected areas (MPAs) are increasingly advocated for the conservation and management of sharks and rays. However, substantial uncertainty remains regarding which species can benefit from MPAs. Meanwhile, area‐focused protection targets have spurred recent and rapid gains in the creation of large MPAs, many of which carry vague objectives set by a diverse group of stakeholders with potentially different notions of “success.” Here, we capture and critically evaluate current views on the use of MPAs for shark and ray conservation. Through interviews with scientists, MPA managers, fisheries experts, conservation practitioners, advocates and policy experts (= 53), we demonstrate a variety of perspectives regarding: (a) the effectiveness of MPAs as a tool for shark and ray conservation; (b) which factors influence the success of MPAs for sharks and rays; and (c) the desired outcomes of these MPAs. While MPAs created specifically for sharks and rays were viewed to be slightly more effective than regular MPAs as a tool for shark and ray conservation, both were generally considered insufficient in isolation. Despite greater emphasis on social success factors (e.g., local support) over biophysical success factors (e.g., size), biological outcomes (e.g., increased abundance) were prioritized over social outcomes (e.g., livelihood benefits). We argue that a stronger focus on achieving social outcomes can enhance the potential for MPAs to benefit sharks and rays. In revealing current thinking regarding the drivers and indicators of MPA success for sharks and rays, the results of this study can inform efforts to conserve and manage these species.  相似文献   

17.
CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) aims to ensure that international trade in specimens of wild animals and plants does not threaten their survival. However, measuring the effectiveness and impacts of these trade regulations for commercially exploited aquatic species remains challenging. This study highlights observed or documented changes in elasmobranch fisheries in eight Southeast Asian countries before and after the listing of sharks and rays in CITES’ Appendix II, and the influence of CITES across five pillars or sectors of a “fishery assessment framework” developed especially for this purpose. Fisheries experts reported change was most common in the “governance” (e.g., policy, regulation and compliance) and “fisher(y)” sectors (e.g., structure and effort) of the assessment framework. The smallest change was recorded in “markets” (e.g., structures and prices) and “sociocultural” sectors (e.g., consumption, livelihoods and community awareness). Overall, the study demonstrates a measurable, albeit small, mostly positive influence of CITES in five of eight countries, while noting predominantly negative influences across two, and ongoing challenges for all in maintaining legal trade of these CITES‐listed species. The study concludes by offering guidance on future needs: most notably, more effort for long‐term collection of fundamental fisher‐, stock‐ and market‐related data to inform adaptive management and facilitation of legal trade where it is shown to be sustainable. Furthermore, as many of the shark and ray species under CITES provisions are transboundary stocks, increased support for communication and cooperation among regional fishery stakeholders is an ongoing need.  相似文献   

18.
Fisheries Science - Tidal flats are ecologically important as they support a large community of animals (e.g., crabs, mollusks, and polychaetes) and restore water quality. However, information...  相似文献   

19.
To clarify the feeding habits of seagrass fishes, we examined the gut contents from 42 fish species collected in seagrass habitats in Trang. Thirteen species showed ontogenetic and/or seasonal changes in food-use patterns. Smaller individuals generally preyed on small planktonic items (e.g., copepod larvae) or small benthic/epiphytic crustaceans (e.g., harpacticoid copepods), with subsequent changes to other prey items (e.g., shrimps, crabs, detritus and filamentous algae) with growth. The most important dietary items for the seagrass fish assemblages comprised benthic/epiphytic crustaceans, detritus, and planktonic copepods. Cluster analysis based on dietary overlaps showed that the seagrass fishes comprised eight feeding guilds (large benthic/epiphytic crustacean, detritus, planktonic animal, small benthic/epiphytic crustacean, mollusc, invertebrate egg, polychaete, and fish feeders). Of these, the first three guilds were the most abundantly represented, whereas the last three were each represented by only a single species.  相似文献   

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