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1.
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
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
2.
鲤疱疹病毒Ⅱ型主要免疫原性蛋白的鉴定 总被引：1，自引：0，他引：1
为了鉴定CyHV-2的主要免疫原性蛋白,本研究用分离的CyHV-2-YC-1分离株(下简称CyHV-2)感染异育银鲫尾鳍细胞系(GiCF),用蔗糖密度梯度超速离心法对细胞感染液中的CyHV-2进行纯化,纯化后的CyHV-2病毒免疫小鼠制备抗CyHV-2多克隆抗体。纯化的病毒颗粒经变性聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝染色后,用抗CyHV-2多克隆抗体进行Western blotting分析和质谱鉴定。结果显示,透射电镜下观察发现50%～66%的蔗糖梯度多见完整囊膜包裹的CyHV-2病毒颗粒,也有少量囊膜破损的病毒颗粒。Western blotting结果显示,抗CyHV-2多克隆抗体与多种病毒蛋白具有特异性免疫反应,质谱鉴定显示,其中8种主要免疫原性蛋白分别是ORF92、ORF115、ORF25、ORF57、ORF66、ORF72、ORF131和ORF132。研究表明,通过蔗糖密度梯度超速离心法提纯CyHV-2病毒颗粒后,本研究制备的抗CyHV-2多克隆抗体能够特异性识别CyHV-2病毒的主要免疫原性蛋白。本研究将为CyHV-2免疫学检测方法的建立以及疫苗的研制提供更多的候选抗原。 相似文献
3.
鲤疱疹病毒3型(Cyprinid herpesvirus 3, CyHV-3)又称为锦鲤疱疹病毒(Koi herpesvirus, KHV),是一种高传染性、致死性病毒。为开发新型CyHV-3 DNA疫苗,前期研究中将CyHV-3 ORF148基因插入pEGFP-N1构建重组质粒,在此基础上,本研究通过转染试验、间接免疫荧光试验证实pORF148-EGFP融合蛋白可以在CCB-J细胞系和建鲤体内表达;将重组质粒作为DNA疫苗,肌肉注射免疫建鲤鱼苗,ELISA检测表明免疫pEGFP-ORF148重组质粒可以显著提高建鲤血清特异性抗体水平; RT-qPCR检测显示,免疫pEGFP-ORF148重组质粒后,建鲤脾脏和头肾中的免疫相关基因如IFN-a1、Mx-1、CXCa、CXCR1、TNF-α、IL-1β及IgM基因表达量均显著提高。攻毒实验显示, CyHV-3攻毒21 d后, PBS组、pEGFP-N1组和pEGFP-ORF148组的建鲤存活率分别为30%、35%和85%,免疫pEGFP-ORF148可以显著提高建鲤的存活率(P0.01)。本研究旨在为CyHV-3 DNA疫苗的应用提供理论依据。 相似文献
4.
鲤疱疹病毒3型T分离株主要免疫原性蛋白的鉴定 总被引：1，自引：0，他引：1
为了鉴定鲤疱疹病毒3型(Cyprinid Herpesvirus 3,CyHV-3)主要免疫原性蛋白,研究采用CyHV-3-T分离株感染CCB细胞系,运用蔗糖密度梯度超速离心方法对CyHV-3进行纯化,纯化的病毒颗粒经SDSPAGE和考马斯亮蓝染色后,用锦鲤(Cyprinus carpio haematopterus)抗CyHV-3阳性血清进行Western blotting分析和液相色谱串联质谱鉴定。结果表明,透射电镜下可观察到大量完整囊膜包裹或只有裸露核衣壳的CyHV-3颗粒,Western blotting结果显示抗CyHV-3阳性血清与多种病毒蛋白具有明显特异性免疫反应,质谱鉴定表明其中4种免疫原性蛋白分别为ORF92、ORF66、ORF72和ORF81,其中ORF66和ORF72为首次鉴定的具有免疫原性衣壳蛋白。本研究将为CyHV-3血清学诊断方法的建立、亚单位疫苗或DNA疫苗的研制提供更多候选抗原。 相似文献
5.
为了对鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)ORF136基因编码蛋白进行功能研究和血清学诊断,本实验通过对ORF136基因推导的第31~157位氨基酸序列进行PCR扩增,并与原核载体pET-32a(+)连接,转化至大肠杆菌Rosetta(DE3)感受态后进行IPTG诱导表达,将纯化后的重组蛋白免疫新西兰白兔(Oryctolagus cuniculus)以制备ORF136多克隆抗体,运用Western blot和间接免疫荧光技术对抗体进行鉴定。结果表明,重组融合表达蛋白大小与预期一致,约为35 kD,且主要分布在包涵体中。Western blot分析显示,免疫兔后获得的纯化ORF136多克隆抗体能特异性识别纯化的CyHV-3和感染CyHV-3的KS细胞;间接免疫荧光分析进一步表明ORF136多抗能识别感染CyHV-3的KS细胞。ORF136多克隆抗体的制备为ORF136蛋白功能研究和CyHV-3血清学诊断方法的建立提供了重要基础。 相似文献
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7.
淇河鲫IL-8原核表达系统的构建及多克隆抗体的制备与鉴定 总被引：1，自引：0，他引：1
为揭示淇河鲫(Carassius auratus var.Qihe)白细胞介素-8(interleukin-8,IL-8)的功能,本研究构建了原核表达系统,并制备了兔抗鲫IL-8多克隆抗体。首先采用RT-PCR法扩增淇河鲫IL-8基因编码序列中不含信号肽的基因片段,克隆到pET-32a(+)载体后转化入Rosetta菌株构建原核表达系统。经IPTG诱导表达出相对分子质量约27.8 kD的目标融合蛋白。将纯化的融合蛋白免疫家兔制备多克隆抗体,效价达1︰10~5。经免疫亲和层析纯化的抗体能特异性识别重组和天然的淇河鲫IL-8蛋白。比较不同组织的免疫组化和荧光定量PCR结果,IL-8蛋白量和mRNA的表达量在不同组织间变化趋势一致,在肌肉、脾和头肾均检测到较高的表达量,肠道的表达量较低。本研究为进一步探讨淇河鲫IL-8的生物学功能奠定了基础。 相似文献
8.
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
9.
基于间接ELISA的鲤血清CyHV-3 pORF65抗体检测方法的建立 总被引：1，自引：0，他引：1
鲤疱疹病毒3型(cyprinid herpesvirus 3,Cy HV-3)囊膜蛋白ORF65基因全长1 785 bp,编码594个氨基酸。该研究在稀有密码子分析及信号肽与跨膜结构预测的基础上,将p ORF65中的N端信号肽与C端跨膜区段删除后进行密码子优化与基因合成,将合成的截短ORF65(truncated ORF65)插入p ET32a(+)载体,构建了p ET32a-trun ORF65;进一步采用DNAstar、ABCpred、Bepi Pred 1.0软件预测了p ORF65的5个B细胞表位优势区段,以合成的截短ORF65为模板,通过SOE-PCR将5个B细胞表位优势区段编码序列融合后插入p ET32a(+)载体,构建了p ET32a-mod ORF65。重组质粒分别转入BL21(DE3)菌株,经IPTG诱导,SDS-PAGE和Western-blot分析,p ET32a-mod ORF65获得高效表达,表达的融合蛋白分子量为56.4 k D。此外,利用r Protein G亲和层析纯化了锦鲤(Cyprinus carpio haematopterus)血清Ig M,免疫小鼠,制备了鼠抗锦鲤Ig M多克隆抗体。在上述研究的基础上,将纯化的p ORF65作为包被抗原,鼠抗锦鲤Ig M多克隆抗体作为检测抗体,建立了间接ELISA方法,该方法可以检测p EGFP-ORF65 DNA疫苗免疫锦鲤后产生的特异性抗体。 相似文献
10.
为研究溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护作用,实验构建了重组真核表达质粒pcDNA-flaC并将该质粒肌肉注射红笛鲷,采用PCR、RT-PCR、ELISA和攻毒试验等方法检测了该真核表达质粒在红笛鲷组织内的分布、表达和对红笛鲷的免疫保护.PCR结果显示,免疫接种7和28 d,注射点周围肌肉、鳃、肾脏、肝脏和脾脏都存在质粒分布;RT-PCR结果显示,免疫接种后第7天、14天和28天,红笛鲷不同组织内均有目的基因表达.ELISA结果表明,鱼血清内产生了抗FlaC蛋白的抗体,表明DNA疫苗免疫后鱼体表达了目的蛋白,并诱导产生了相应抗体.攻毒实验表明,免疫后的红笛鲷能较好地抵抗致病性溶藻弧菌的感染.结果表明,质粒pcDNA-flaC可能是抵抗溶藻弧菌感染的有效的疫苗候选物. 相似文献
11.
一种基于Ⅱ型鲤疱疹病毒衣壳蛋白72的免疫学检测方法 总被引：4，自引：1，他引：3
Ⅱ型鲤疱疹病毒(cyprinid herpesvirus 2,Cy HV-2)是引起养殖异育银鲫(Carassius auratus gibelio)造血器官坏死症的致病病原。在临床筛查中基于病毒核酸的PCR和real time PCR技术已经建立,但是稳定性更强的免疫学诊断技术国内外尚无报道。本研究目的是利用Cy HV-2编码的ORF72基因(Gen Bank登录号:AFJ20502.1)所编码的衣壳蛋白作为捕获抗原,通过识别感染病毒的鱼体中的相应抗体,从而对样本进行临床免疫学检测。首先采用PCR方法从纯化的Cy HV-2基因组中扩增ORF72基因,并把该基因克隆至原核表达载体PGEX-4T-3,并转化到大肠杆菌中诱导表达,诱导表达的产物通过SDS-PAGE进行鉴定,对表达的重组蛋白进行纯化。用已纯化的72重组蛋白对小鼠进行免疫,制得72重组蛋白的抗体。Western blot检测表明所制备的多克隆抗体既能识别原核表达的重组蛋白,也可以识别Cy HV-2病毒粒子上的衣壳蛋白72。在上述基础上建立了基于Western blot技术的Cy HV-2抗体检测技术:用纯化的72重组蛋白作为检测抗原,鲫鱼血清用作一抗,兔抗鲫Ig M多克隆抗体作为二抗,酶标羊抗兔作为三抗鉴定鲫鱼是否存在Cy HV-2特异性抗体。在对急性感染期的临床样本检测中,本方法能在所有样本中检测出ORF72特异性抗体存在,表明72重组蛋白作为相应抗体捕获原可以用于确诊鲫鱼是否感染Cy HV-2。本研究建立的实验室免疫学检测方法为商品化免疫学检测技术的开发奠定了基础,对Cy HV-2的检验检疫具有一定的临床应用价值。 相似文献
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通过体外重组，将绿色荧光蛋白基因编码序列克隆到鲤鱼肌动蛋白基因启动子下游，构建成能在鱼体内表达绿色荧光蛋白的重组分子。用限制性内切酶PvuI酶切线性化后，通过显微注射法导入金鱼受精卵内，在转化后48h，可观察到绿色荧光，经PCR初步筛选后，对显阳性个体进行Southern杂交检测，结果表明，转化个体表现出较强的杂交信号。这说明绿色荧光蛋白基因能在金鱼体内整合、表达。 相似文献
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Responses to fishmeal and soybean meal‐based diets by three kinds of larval carps of different food habits 
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下载免费PDF全文 A 60‐day trial was conducted in a recirculation system to determine the responses of herbivorous grass carp (Ctenopharyngodon idellus) (0.43 g), omnivorous gibel carp (Carassius auratus gibelio CAS III) (0.46 g) and carnivorous black carp (Mylopharyngodon piceus) (0.46 g) when fed with fishmeal (FM)‐ or soybean meal (SBM)‐based diets. Two isonitrogenous (380 g kg?1 DM), isolipid (80 g kg?1 DM), isocaloric (17.75 kJ g?1) diets were formulated using FM and SBM as main protein sources. The results showed that weight gain (WG), feed efficiency (FE) and protein retention efficiency (PRE) in FM group were significantly higher than those in SBM group. Grass carp showed better utilization on SBM than other two species and presented higher WG, FE and PRE. When fed with SBM diet, intestinal chymotrypsin (CHY), lipase and trypsin (TRY) activities of gibel carp and black carp decreased while higher α‐amylase (α‐AMY) activity of grass carp increased. An enteritis‐like effect was observed in black carp and grass carp fed SBM diet. Intestine bacteria mainly belonged to Firmicutes in gibel carp and Proteobacteria in black carp. The dominant genera identified in three species were Cellulomonas, belonging to Actinobacteria. In conclusion, SBM decreased the growth of three carps. Grass carp was more adapted to plant protein. 相似文献
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Effect of substitution of dietary fishmeal by soya bean meal on different sizes of gibel carp (Carassius auratus gibelio): nutrient digestibility,growth performance,body composition and morphometry 下载免费PDF全文
下载免费PDF全文 Four 70‐day growth trials were performed to investigate the effects of substitution of dietary fishmeal (FM) by soya bean meal (SBM) in different sizes of gibel carp CASIII (Carassius auratus gibelio). The initial weights of fry, juvenile, 1‐year fish and broodstock of gibel carp were 0.8, 5.0, 62.7 and 135.6 g, respectively. In each trial, eight diets were formulated to be iso‐nitrogenous and iso‐energetic, and different levels of soy bean meal protein were used to replace fishmeal protein at 0%, 20%, 40%, 60%, 80% and 100%. Another diet containing all SBM protein and supplemented with crystalline amino acids was also tested. The results showed that apparent digestibility coefficients (ADCs) of dry matter and energy decreased with SBM substitution while the ADCs of phosphorus increased. The ADCs of protein of high SBM substitution were higher than those of FM groups. Juveniles show lower ADCs than grown‐up fish. Dietary SBM substitution showed negative effects on survival, growth and feed utilization in gibel carp. One‐year fish and broodstock gibel carp have relatively higher tolerance to dietary SBM than fry and juveniles. 相似文献
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A 74‐day experiment was conducted to evaluate the production performance and water quality variation in three types of farming system for largemouth bass Micropterus salmoides. The tested aquaculture models included monoculture of largemouth bass (MC), polyculture of largemouth bass, gibel carp Carassius auratus gibelio and silver carp Hypophthalmichthys molitrix (PC), and integrated culture of largemouth bass, gibel carp, silver carp and freshwater pearl mussel Hyriopsis cumingii (IC). The ratio of largemouth bass, gibel carp and silver carp was 30:2:1 in the PC model, and the ratio of largemouth bass, gibel carp, silver carp and mussel was 30:2:1:5 in the IC model. The largemouth bass were fed with formulated feed twice daily. No significant differences were found in weight gain and yield of largemouth bass, total fish yield, nitrogen (N) and phosphorus (P) utilization efficiencies, N and P wastes, pH, nitrite, nitrate, reactive phosphate, total nitrogen, total phosphorus, total organic carbon, chemical oxygen demand, 5‐day biochemical oxygen demand, chlorophyll a, primary productivity among the MC, PC and IC models. The ammonia was lower, while the dissolved oxygen was higher in the PC tanks than in the MC tanks. These results suggest that the environment situation was better in the PC tanks relative to that in the MC tanks. The present study reveals that the PC model should be a way to optimize the aquaculture model for commercial largemouth bass farming. 相似文献
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Detection methods of Cyprinid herpesvirus 2 infection in silver crucian carp (Carassius auratus gibelio) via a pORF72 monoclonal antibody 下载免费PDF全文
下载免费PDF全文 Cyprinid herpesvirus 2 (CyHV‐2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid‐based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV‐2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV‐2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV‐2. First, ORF72 gene was amplified from the CyHV‐2 genome and cloned into a PGEX‐4t‐3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV‐2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio. 相似文献
17.
Carbohydrate utilization by herbivorous and omnivorous freshwater fish species: a comparative study on gibel carp (Carassius auratus gibelio. var CAS III) and grass carp (Ctenopharyngodon idellus) 下载免费PDF全文
下载免费PDF全文 Xiangsong Li Xiaoming Zhu Dong Han Yunxia Yang Junyan Jin Shouqi Xie 《Aquaculture Research》2016,47(1):128-139
Two 8‐week feeding trials were conducted to evaluate dietary carbohydrate utilization by omnivorous gibel carp (Carassius auratus gibelio) (2.4 ± 0.1 g) and herbivorous grass carp (Ctenopharyngodon idellus) (6.5 ± 0.1 g). Five isonitrogenous (370 g kg?1) and isolipid (70 g kg?1) diets were formulated with increasing corn starch levels (60, 140, 220, 300 and 380 g kg?1). Results showed that specific growth rate (SGR), feed efficiency (FE) and protein retention efficiency (PRE) of gibel carp significantly increased from dietary starch of 60 to 300 g kg?1 and then decreased from 300 to 380 g kg?1, but those of grass carp showed no significant differences between treatments. Independent of dietary starch levels, grass carp gained significantly higher FE and PRE than gibel carp. Feeding rate (FR) of gibel carp was significantly higher than that of grass carp. In two fish species, high dietary starch (300 and 380 g kg?1) tended to obtain higher hepatosomatic index (HSI), serum triglyceride, hepatic lipid and body lipid contents. Serum glucose concentration of grass carp was not affected, while that of gibel carp fed the starch of 300 g kg?1 diet was significantly lower than those of the fish fed other four diets (60, 140, 220 and 380 g kg?1). Grass carp showed high tolerance to dietary starch while dietary corn starch should be no more than 300 g kg?1 for gibel carp. High starch contents may cause lipid accumulation in the liver and body. 相似文献