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自从1971年Engvall等发表了酶联免疫吸附测定(enzyme linked i mmunosorbent as-say,ELISA)用于IgG定量测定的文章后,使1986年开始用于抗原定位的酶标抗体技术发展成液体标本中微量物质的测量方法,即当今广泛使用的ELISA技术。酶联免疫吸附分析法是把抗原抗体的免疫反应和酶的高效催化作用原理有机地结合起来的一种检测技术。该技术主要的依据有三点:第一、抗原(抗体)能结合到固相载体的表面仍具有其免疫活性;第二、抗体(抗原)与酶结合所形成的结合物仍保持免疫活性和酶的活性;第三,结合物与相应的抗体(抗原)反应后,结合的酶仍能催化… 相似文献
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单克隆抗体作为一种具有靶向性的生物大分子,始终是基础医学工作者研究的热点之一。临床上,已被用于治疗肿瘤、病毒感染、自身免疫病、中毒性休克、RH溶血症和抗移植排斥等。但单克隆抗体的临床应用受到一定限制,主要障碍是诱导产生人抗鼠抗体(HAHA反应)、肿瘤渗入量低、亲和力低和半衰期短等。然而,近年来分子生物学技术的发展和向各学科的渗透,人们已经可以通过基因操作技术对抗体进行改造,使其适用于疾病的治疗。如近年来应用噬菌体抗体库技术(phagedisplayantibodylibrarytechniques),筛选具有特异治疗作用的人源性单克隆抗体。另外,通过与基因敲除(knockout)和置换(knockin)技术结合筛选获得完全人源化的单克隆抗体也备受人们的普遍关注。本文就这些技术的基本原理、特点和治疗性抗体的研究进展进行了介绍。 相似文献
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通过制备仿刺参补体C3(AjC3)多克隆抗体,为进一步研究仿刺参补体AjC3免疫机制奠定基础。利用PCR技术扩增AjC3部分基因片段(4556~5110bp),将该片段与原核表达载体pGS-21a连接。将重组表达质粒转化到Transetta(DE3)中经IPTG诱导表达。表达的重组蛋白经镍柱纯化后,作为抗原免疫小鼠制备AjC3多克隆抗体。分别用间接ELISA,Western blot检测抗体的效价和特异性。结果显示,PCR扩增得到约555bp的目的片段,重组蛋白分子量大小约56ku;间接ELISA检测抗体效价达1∶25600,Western blot结果显示,多克隆抗体具有良好的特异性。该试验成功的制备了补体AjC3多克隆抗体,为补体AjC3的进一步研究提供了检测工具。 相似文献
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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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为了解黄颡鱼IgM基因表达的个体发生和IgM抗体代间传递机制,实验利用Real-time PCR和ELISA等技术研究了黄颡鱼IgM重链基因在卵巢、胚胎和仔鱼的表达变化,以及黄颡鱼IgM抗体在卵巢、胚胎和仔鱼中的含量变化。结果显示,黄颡鱼3~7 d仔鱼中,没有检测到IgM mRNA;14 d仔鱼IgM基因开始表达。用细菌免疫亲鱼后,对仔鱼IgM mRNA表达没有影响。黄颡鱼IgM抗体在卵巢、胚胎和仔鱼中都有分布,且呈现下降趋势,至9 d仔鱼中抗体水平最低(是卵抗体含量的0.31倍)。14 d仔鱼中IgM抗体水平上升(是9 d仔鱼抗体含量的1.6倍)。用细菌免疫亲鱼后,能显著提高胚胎、仔鱼中的IgM抗体水平,在卵中抗体含量提高了2.3倍,9 d仔鱼中提高了1.8倍。研究表明,黄颡鱼IgM抗体可以在母本和后代之间传递,早期仔鱼IgM抗体主要来自于亲本;因此,免疫亲鱼能显著增加子代的抗体水平。 相似文献
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病毒性出血性败血症病毒单链抗体的原核表达及其功能鉴定 总被引：1，自引：1，他引：0
为制备病毒性出血性败血症病毒(VHSV)单链抗体(single chain variable fragment antibody,ScFv)并鉴定其生物学功能,本研究提取抗VHSV单抗1G5的杂交瘤细胞株总RNA并反转录获得cDNA模板,通过PCR扩增VHSV抗体的轻链可变区(VL)和重链可变区(VH)编码序列,将其拼接成单链抗体Sc Fv基因后插入载体pET28a中,构建原核表达重组质粒并在大肠杆菌中诱导表达。结果表明,单链抗体主要以可溶性形式表达,分子量约28 ku,能特异性识别VHSV病毒的G蛋白并对VHSV病毒具有体外中和活性,其对VHSV病毒的G蛋白亲和力(KD)达到1.4×10~(–8) M。单链抗体ScFv的制备为进一步研究VHSV的治疗性抗体、快速诊断试剂奠定了基础。 相似文献
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嗜水气单胞菌灭活疫苗浸泡后鳜IgM基因表达量和抗体效价的变化 总被引：3，自引：2，他引：1
应用荧光定量PCR技术,研究了嗜水气单胞菌疫苗浸泡免疫后,鳜(Siniperca chuatsi)鳃、皮肤、脾脏和头肾中IgM基因表达量变化,同时应用ELISA检测皮肤黏液和血清中抗体滴度变化。结果显示:最早检测到IgMmRNA转录水平上调的是皮肤和鳃(第4天),而脾脏和头肾在第7天才达到高峰。IgM基因在头肾和脾脏中表达量较高(高峰值分别达到16.3和23.8),皮肤和鳃中的表达量较小(高峰值分别为4.3和8.6)。抗体效价方面,皮肤黏液中抗体滴度峰值出现时间较早(第7天),但抗体从开始形成到消失持续时间较短(28 d);血清中抗体滴度峰值出现时间较迟(第21天),但持续较长(42 d)。结果表明:浸泡免疫能够引发鳜机体和局部均出现免疫应答。从抗体滴度和IgM基因表达量方面考虑,系统免疫组织均高于黏膜免疫组织,表明前者是合成IgM的主要场所。从时间上比较,对抗原刺激最早做出应答的是皮肤黏膜和鳃,其后系统免疫才表现应答作用,这表明了局部黏膜能够在抗原入侵的早期起到抵制作用。 相似文献
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A combined phage display ScFv library against Myxobolus rotundus infecting crucian carp, Carassius auratus auratus (L.), in China 总被引：1，自引：0，他引：1
Immunological methods have been developed for the diagnosis of Myxobolus rotundus but their use has been limited for the prevention and therapy of this serious parasitic pathogen. Phage display antibody libraries are a powerful technique for the development of antibodies to molecules of interest and have advantages over traditional hybridroma approaches. In the present study, four antigen fractions related to M. rotundus were prepared and a combined phage display single-chain antibody fragments (ScFv) library was constructed against this parasite. Preliminary analysis indicated that a combined antibody library of about 2.08 x 10(5) individual clones and high diversity was generated. After four rounds of screening (bio-panning) against soluble spore protein prepared from lysed, intact, mature M. rotundus spores, a strain monoclonal phage display ScFv, termed pCAN-6H9, with better affinity, was isolated. The pCAN-6H9 gene fragment was sequenced and analysed. The specificity of pCAN-6H9 was further demonstrated by dot-blot. In competition enzyme-linked immunosorbent assay, both the original and enriched phage-displayed ScFv repertoire showed significant inhibition of mouse anti-M. rotundus serum binding to coated antigen, while the inhibition rate of monoclonal pCAN-6H9 phage particles was only 11.83%. 相似文献
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水产生物技术研究的回顾、最新进展及前景展望 总被引：2，自引：0，他引：2
1简要回顾水产生物技术是20世纪80年代开始发展起来的、以水产生物为主要研究对象,以水产业应用为目的,以基因工程、细胞工程、发酵工程、酶工程等现代生物技术为主体的综合性技术体系。水产生物技术是生物技术的重要组成部分,也是当今 相似文献
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鱼类生殖细胞移植技术是通过诱导不同物种之间生殖系嵌合体来实现。原始生殖细胞和精原细胞(或卵原细胞)是诱导生殖细胞系嵌合体的关键材料。在过去的十多年中,通过采用不同的供体生殖细胞和不同发育阶段(囊胚期胚胎、初孵仔鱼和成鱼)的鱼为受体,开发出多种鱼类生殖细胞移植技术。这些成果的取得,为生殖细胞移植技术应用于诸多水产养殖新兴领域打下了坚实基础。该技术可以应用于:(1)生殖细胞生物学和转基因鱼等基础研究;(2)遗传资源和濒危鱼种的保护;(3)鱼类性别选择育种;(4)有效提高放流鱼苗的遗传多样性。 相似文献
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Angelisa T.Y. Osmond Stefanie M. Colombo 《Journal of the World Aquaculture Society》2019,50(3):490-509
Advancements in gene technology in recent years have been driving the aquaculture industry forward. Improvements in growth performance, feed efficiency, and omega‐3 content are goals of the industry that could capitalize on applications of genetic engineering. One of the major challenges in the industry is to reduce the use of fish meal and oil, to improve the environmental and economic sustainability of aquaculture. The recent development of genetically engineered feed ingredients is one potential solution to the looming problem of fish meal and oil dependency. Furthermore, the development of transgenic fish has potential to improve production efficiency and other future desirable characteristics that relate to feed utilization and product quality. New gene technologies are beginning to revolutionize how we produce our food, and in aquaculture, will ultimately reduce pressure on wild fish stocks, help to preserve natural aquatic ecosystems, and improve nutritional profiles of farmed fish for human consumption. The purpose of this review is to provide an update on the current applications of genetic engineering technology to improve aquaculture through nutrition, including the development and use of transgenic feed ingredients, transgenic fish, and ultimately their impacts on nutrition, product quality, and consumers. 相似文献
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Siau-Hoi Lim Hui-Ting Ho Syed Musthaq S. Khader Jimmy Kwang 《Aquaculture (Amsterdam, Netherlands)》2009,295(1-2):134-137
As guppies are one of the commercially important fish in freshwater ornamental aquaculture industry, it is important to gain an understanding of guppy immune response for infectious disease control. Till now, the number of study that examined the immune response of guppies is limited and effective tools for monitoring guppy antibody have not been reported. In this study, we successfully isolated guppy IgM using mannan-binding protein (MBP) affinity chromatography and produced specific polyclonal antibodies against guppy IgM heavy and light chains, that showed a molecular weight of approximately 74 and 23 kDa respectively. The produced polyclonal antibodies were used to develop an enzyme-linked immunosorbent assay (ELISA) and have demonstrated to be an effective tool for the detection and quantification of antigen-specific antibody of guppies immunized with Pseudomonas fluorescens. In conclusion, the produced anti-guppy IgM polyclonal antibodies should prove its future implications for immunology and epidemiology studies in guppies. 相似文献
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Roselien Crab Yoram Avnimelech Tom Defoirdt Peter Bossier Willy Verstraete 《Aquaculture (Amsterdam, Netherlands)》2007,270(1-4):1-14
As the aquaculture industry intensively develops, its environmental impact increases. Discharges from aquaculture deteriorate the receiving environment and the need for fishmeal and fish oil for fish feed production increases. Rotating biological contactors, trickling filters, bead filters and fluidized sand biofilters are conventionally used in intensive aquaculture systems to remove nitrogen from culture water. Besides these conventional water treatment systems, there are other possible modi operandi to recycle aquaculture water and simultaneously produce fish feed. These double-purpose techniques are the periphyton treatment technique, which is applicable to extensive systems, and the proteinaceous bio-flocs technology, which can be used in extensive as well as in intensive systems. In addition to maintenance of good water quality, both techniques provide an inexpensive feed source and a higher efficiency of nutrient conversion of feed. The bio-flocs technology has the advantage over the other techniques that it is relatively inexpensive; this makes it an economically viable approach for sustainable aquaculture. 相似文献
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本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein,GFP)基因的重组质粒(smyd1:gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1:gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。 相似文献
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Heidi Mikkelsen Thomas Lindenstrøm Michael Engelbrecht Nielsen 《Journal of the World Aquaculture Society》2006,37(4):518-522
Abstract.— The effect of temperature on production and affinity of antibodies against antigens from the parasitic ciliate Ichthyophthirius multifiliis were studied in rainbow trout ( Oncorhynchus mykiss ). Fish were immunized with I. multifiliis antigens and reared at three different temperatures, 5, 12, and 20 C for 56 d. The production of specific antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that temperature has a pronounced effect on the assay result of the ELISA technique. Plasma samples analyzed at 5 C showed that the specific antibody response of fish reared at 5 C was similar to fish reared at 12 and 20 C. However, when samples were assayed at 12 and 20 C, the measured antibody response tended to be higher for the samples from trout reared at 12 and 20 C. Additionally, it was found that rainbow trout reared at 5 C showed a delayed but not hampered antibody response compared to fish reared at 12 and 20 C, indicating a correlation between low temperature and delayed antibody production. 相似文献
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Within aquaculture, genetic engineering (GE) is emerging as a powerful method for breeding of fish and shellfish, and for developing alternative sources of feed and vaccines to combat diseases. On the other hand, the use of GE in aquaculture raises ecological, ethical and economic concerns. For instance, genetically modified (GM) feed could be spread to the aquatic environment and consumed by other marine organisms, and horizontal gene transfer may conceivably occur from DNA in feed or vaccines to a recipient genome or by faeces to the environment. Numerous reports have described beneficial effects such as viral disease resistance following DNA vaccination. However, side effects, such as activation of other genes than those which are central in immune defence mechanisms, may occur and warrant further investigations. In order to achieve sustainable introduction of GE, it is crucial that appropriate scientific investigations and ethical considerations are done prior to large-scale introduction of GE products such as DNA/GE vaccines and GM feed in commercial fish farming. This may result in a solid basis for the avoidance of potentially undesirable health and environmental effects. If GE can help make aquaculture a sustainable industry, this opens the possibility of positive market and consumer responses. This can best be achieved by involving the stakeholders from the conceptual stage to the commercial stage by facilitating a transparent process whose purpose is to inform research, to identify decision stakes, and to influence design, adoption and implementation of pro-active policy. 相似文献