共查询到19条相似文献，搜索用时 187 毫秒
1.
马白细胞介素-18单克隆抗体的制备及其特性的初步鉴定 总被引：1，自引：0，他引：1
摘要：利用纯化的用原核系统表达的重组马白细胞介素-18成熟蛋白(pET-mEIL-18)抗原免疫BALB/c 小鼠，取脾细胞与SP2/0骨髓瘤细胞进行融合，同时以昆虫杆状病毒表达系统获得的马白细胞介素-18成熟蛋白(mEIL-18)作为抗原进行间接ELISA检测，筛选并最终得到了9株能稳定分泌抗体的单细胞克隆株。利用IFA试验对9株单克隆抗体进行鉴定，结果证实所获得的9株细胞分泌的抗体都是针对mEIL-18的特异性抗体。将此9株单克隆抗体分别与用原核系统分段表达的重组马白细胞介素-18成熟蛋白pET-mEIL-18-(1)(mEIL-18 1～78位氨基酸残基)和pET-mEIL-18-(2)(mEIL-18 79～157位氨基酸残基)蛋白进行特异性ELISA检测，结果表明，9株单克隆抗体有5株识别表达的pET-mEIL-18-(1)(mEIL-18 1～78位氨基酸残基)，而另外4株单克隆抗体与pET-mEIL-18-(2)(mEIL-18 79～157位氨基酸残基)发生反应，说明所获得的9株单克隆抗体至少针对pET-mEIL-18 2个或2个以上不同的抗原表位。单克隆抗体亚型鉴定试剂盒的鉴定结果显示，除M1F11、M3A11和N7D9为IgM，其余的单克隆抗体均为IgG1，而且所有单克隆抗体的轻链均为?资链。 相似文献
2.
热激蛋白90家族(heat shock protein 90(HSP)family)是一种进化保守的蛋白质,在分子伴侣功能、细胞循环调控、抗原传递方面发挥着重要作用。本研究用RT-PCR和RACE技术,克隆得到了松材线虫(Bursaphelenchus xylophilus)、拟松材线虫(Bursaphelenchus mucronatus)和豆伞滑刃线虫(Bursaphelenchus doui)热激蛋白90基因的cDNA全长序列,分别命名为Bx-hsp90、Bm-hsp90和Bd-hsp90,(GenBank登录号分别为:GU013561、HMO34943和HM347331),其cDNA全长分别为2255、2193和2232bp,开放阅读框均为2127bp,编码708个氨基酸,5'端非编码区的长度分别为33、13和13bp,3'端非编码区的长度分别为95、53和92bp。其DNA全长分别为2440、2388和2407bp,包含3个内含子。三者氨基酸序列的一致性为98.78%。进化分析表明,松材线虫、拟松材线虫、豆伞滑刃线虫与秀丽小杆线虫(Caenorhabditis elegans)的亲缘关系较近,推测三... 相似文献
3.
松材线虫病致病机理的研究进展 总被引：1，自引：0，他引：1
松材线虫病是一种为害林业生态环境的主要虫害。随着松材线虫致病机理的研究不断深化,人们逐渐了解到出现松材线虫病的病原是松材线虫和细菌,其严重威胁森林的正常生长,破坏林业生态环境。松材线虫内部的酶会侵蚀树木的薄壁细胞,树脂不正常的渗透,管胞内部水分传输受到阻碍,树木枯萎。如果松树感染松材线虫病,物质进入到松树管胞中会形成空洞,严重情况下导致树木死亡。基于此,对松材线虫病致病机理研究进展进行分析,以求为后续的工作提供参考。 相似文献
4.
松材线虫与拟松材线虫rDNA中ITS区的比较研究 总被引：18，自引：0，他引：18
本研究利用PCR－RFLP和DNA测序技术分析了松材线虫和拟松材线虫rDNA中的ITS区。ITS区界于rDNA的18S和28S基因之间,并被5.8S基因分为ITS1和ITS2两个区。5种限制性内切酶酶切结果表明,松材线虫和拟松材线虫rDNA的ITS区存在着显著的差异;在松材线虫不同来源株系中,来自中国和日本的株系的酶切图谱一致,与来自加拿大的株系有着微小差别。对南京和日本的松材线虫及富阳的拟松材线虫株系ITS1区的序列分析显示在ITS1的280bp中,南京和日本的松材线虫株系之间,同源性高达99.7%,而来自富阳的拟松材线虫与南京和日本的松材线虫ITS1区序列的同源性相对较低,分别为90％和89.6%。 相似文献
5.
制备兔多杀性巴氏杆菌(Pasteurella multocida,Pm)外膜蛋白A(OmpA)重组蛋白单抗,为兔多杀性巴氏杆菌病的诊断提供特异性强,灵敏度高的单克隆抗体。本研究选取并扩增Pm外膜蛋白基因OmpA,原核表达获得了的大小为37.6kD的OmpA重组蛋白,以纯化复性的兔多杀性巴氏杆菌OmpA重组蛋白作为免疫原,按常规方法免疫BALB/c小鼠(Mus musculus),取其脾细胞与SP2/0细胞融合,ELISA筛选出了4株分泌Pm OmpA蛋白单克隆抗体的杂交瘤细胞,分别命名为5D2、BC11、2A2和6A4。其中2A2细胞培养液效价为1∶256,5D2、BC11和6A4效价为1∶128;2A2腹水效价为1∶12800,其余3株达1∶6400。杂交瘤细胞经反复冻存、复苏及多次传代,仍能稳定分泌高效价抗体。Western blot显示,4株单克隆抗体均能与Pm OmpA重组蛋白重组发生特异性反应。用ELISA方法鉴定2A2单克隆抗体亚型为IgG2b,Protein A亲和纯化2A2腹水抗体,获得的单抗浓度为130μg/mL。选取纯化的2A2单克隆抗体进行潜在应用研究,Western blot与间接免疫荧光结果显示,单克隆抗体能与兔多杀性巴氏杆菌分离菌株发生特异性反应。表明利用体外重组表达兔多杀性巴氏杆菌OmpA融合蛋白制成的杂交瘤能够稳定分泌效价高、特异性强的单克隆抗体,可用于兔多杀性巴氏杆菌诊断,本研究结果为兔多杀性巴氏杆菌诊断试剂盒的研制提供技术参数。 相似文献
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为制备可以区分牛病毒性腹泻1型病毒(BVDV1)和2型病毒(BVDV2)的单克隆抗体,利用纯化的BVDV1和BVDV2为抗原免疫BALB/c小鼠(Mus musculus),取脾细胞与骨髓瘤细胞SP2/0融合,同时以BVDV1和BVDV2病毒作为包被抗原进行间接BVDV1-ELISA和BVDV2-ELISA检测,获得了3株单克隆抗体,其中1株是针对BVDV1的特异性抗体命名为BV1,另外1株是针对BVDV2的特异性抗体命名为BV2,第3株与BVDV1和BVDV2病毒都反应命名为BV12.交叉实验表明,3株单抗中仅有BV12与猪瘟病毒(Hog chorera virus,HCV)反应.BV1、BV2和BV12单抗腹水效价分别为106、107和106,均具有中和活性,中和效价最高达1:524288,分别属于鼠IgG1、IgG2a和IgG2a亚类,轻链均为κ链.3株杂交瘤细胞的染色体数目分别为102、99和100条.本研究获得3株单克隆抗体可以区分BVDV1和BVDV2的杂交瘤细胞. 相似文献
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摘要：将原核表达的重组马-干扰素成熟蛋白（mEIFN-）免疫BALB/c小鼠,经4次免疫后,取脾细胞与SP2/0骨髓瘤细胞进行融合,以昆虫杆状病毒表达系统获得的重组马-干扰素作为检测抗原进行间接ELISA检测,共筛选到15个阳性细胞株。分别经过3轮亚克隆后,最终得到了12株能稳定分泌抗体的单细胞克隆株。利用间接免疫荧光（IFA）实验对12株单克隆抗体进行鉴定,结果证实所获得的12株细胞分泌的抗体均为针对马-干扰素的特异性抗体。单克隆抗体亚型鉴定结果显示,4株为IgG1、2株为IgG2a、3株为IgG2b、3株为IgM,而且所有单克隆抗体的轻链均为链。将此12株单克隆抗体分别与用原核系统分节段表达的重组马-干扰素GST-mEIFN-(1-84)、GST-mEIFN-(80-146)蛋白进行特异性ELISA检测,结果表明有7株单克隆抗体识别GST-mEIFN-(1-84),而另5株单克隆抗体与GST-mEIFN-(80-146)发生反应,说明所获得的12株单克隆抗体至少针对两个或两个以上不同的抗原表位。这将为今后建立马-干扰素检测方法、监测机体免疫状态和研究机体免疫机制奠定了基础。 相似文献
8.
以纯化的原核表达的鸡贫血病毒结构蛋白为抗原，免疫6-8w的雌性Balb/c鼠, 经过三次免疫后，取其脾细胞与SP2/0骨髓瘤细胞进行融合，以纯化的蛋白为抗原进行ELISA检测，将阳性细胞孔经过三次有限稀释，共获得6株能稳定分泌特异性抗体的阳性细胞株。间接免疫荧光试验证实6株单抗可以与鸡贫血病毒感染的MDCC-MSB1细胞发生特异性反应，Western blot结果显示单抗与杆状病毒表达的VP1重组蛋白可以发生特异性反应。利用单克隆抗体亚型鉴定试剂盒进行亚型鉴定，结果表明6株单抗均为IgG1亚型，且所有单抗的轻链均为κ链。用单克隆抗体对分段表达的VP1蛋白进行免疫印迹分析，初步鉴定了筛选出的单抗所对应的抗原表位，其中有四株单抗识别的表位位于VP1 218-274位氨基酸之间，另外两株单抗识别的表位分别位于275-301位、324-369位氨基酸之间。 相似文献
9.
人工雌性激素己烯雌酚单克隆抗体的制备及表征 总被引：1，自引：0，他引：1
合成了己烯雌酚的半抗原CP-DES,并将半抗原与牛血清蛋白(BSA)和卵清蛋白(OVA)共价偶联制备免疫原和包被原。将免疫好的Balb/c小鼠脾脏细胞和SP2/0骨髓瘤细胞融合,筛选出了1株能稳定分泌己烯雌酚单克隆抗体的杂交瘤细胞株2D87C412C7。分析该杂交瘤细胞的染色体,并以间接酶联免疫吸附分析法(iELISA)检测了单克隆抗体免疫球蛋白的类型。获得的杂交瘤细胞的平均染色体数目为45-50对左右,它分泌IgG1亚类的单克隆抗体。该细胞株体外传代和冻存复苏后抗体分泌稳定,细胞上清效价大于640,诱导腹水效价大于108,该单克隆抗体的检测限IC20=2.3ng/ml,与己烯雌酚结构类似物存在一定的交叉反应,与两种载体蛋白(BSA,OVA)无交叉反应。本研究为己烯雌酚ELISA检测方法的建立提供了条件。 相似文献
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猪繁殖与呼吸综合征(porcine reproductive and resp iratory syndrom e,PRRS)是以妊娠母猪繁殖失败和仔猪出现呼吸困难为特征的传染病,PRRSV是该病的病原体。将PRRSV CH-1a株ORF6基因疏水性较强的区域删除后,克隆于pGEX-6p-1载体中,转化大肠杆菌,并进行诱导表达。经SDS-PAGA分析发现,表达了M r约为37 000的融合蛋白rtM,W estern b lot分析证实,重组蛋白能被PRRSV抗血清所识别。收获融合表达的rtM,按50μg/只的剂量与等量弗氏佐剂乳化后,经腹腔接种BALB/c小鼠,免疫3次后,取其脾细胞与SP2/0骨髓瘤细胞进行融合,用rtM作为包被抗原,通过间接EL ISA方法筛选阳性克隆,结果获得了1株能稳定分泌抗rtM蛋白抗体的杂交瘤细胞株,将其命名为M 2B3。间接免疫荧光检测结果发现,所获得的单克隆抗体能与PRRSV感染细胞产生特异性免疫荧光。亚型鉴定结果显示,单克隆抗体M 2B3为IgG 1型,其轻链均为κ链。研究获得的融合蛋白rtM及单克隆抗体将为今后深入研究PRRSV病毒结构和功能,分析M蛋白的抗原表位等提供有益帮助。 相似文献
11.
Kim J Seo SM Lee SG Shin SC Park IK 《Journal of agricultural and food chemistry》2008,56(16):7316-7320
Commercial essential oils from 28 plant species were tested for their nematicidal activities against the pine wood nematode, Bursaphelenchus xylophilus. Good nematicidal activity against B. xylophilus was achieved with essential oils of coriander (Coriandrum sativum), Oriental sweetgum (Liquidambar orientalis), and valerian (Valeriana wallichii). Analysis by gas chromatography-mass spectrometry led to the identification of 26, 11, and 4 major compounds from coriander (Coriandrum sativum), Oriental sweetgum (Liquidambar orientalis), and valerian (Valeriana wallichii) oils, respectively. Compounds from each plant essential oil were tested individually for their nematicidal activities against the pine wood nematode. Among the compounds, benzaldehyde, trans-cinnamyl alcohol, cis-asarone, octanal, nonanal, decanal, trans-2-decenal, undecanal, dodecanal, decanol, and trans-2-decen-1-ol showed strong nematicidal activity. The essential oils described herein merit further study as potential nematicides against the pine wood nematode. 相似文献
12.
Production of a monoclonal antibody against ochratoxin A and its application to immunochromatographic assay 总被引：3，自引：0，他引：3
Cho YJ Lee DH Kim DO Min WK Bong KT Lee GG Seo JH 《Journal of agricultural and food chemistry》2005,53(22):8447-8451
A monoclonal antibody (Mab) against ochratoxin A (OTA) was produced from the hybridoma cell line C7G25, which was established by the fusion of Sp2/0-Ag14 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with the OTA-bovine serum albumin conjugate. This Mab belongs to the IgG(2a) heavy-chain subclass with a kappa-type light chain. The level of 50% inhibition concentration was 1.20 ng/mL in a competitive direct enzyme-linked immunosorbent assay (cdELISA), and the detection limit was 0.12 ng/mL. This antibody is specific for OTA but also shows cross-reactivity with ochratoxin B (31.7%) in a cdELISA. On the basis of the sandwich format using the produced Mab against OTA, a rapid immunochromatographic assay was developed to efficiently detect OTA. This method was able to detect up to 500 ng/mL of OTA in <10 min. 相似文献
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A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the analysis of gossypol in cottonseed meals. First, the checkerboard method was used to determine the optimum amount of coating antigen gossypol-BSA (bovine serum albumin) and primary anti-gossypol monoclonal antibody (Mab) needed in the ic-ELISA. Second, the effects of several physical (incubation time and temperature) and chemical (solvent types and concentrations) conditions on the performance of Mab on ic-ELISA were investigated to get a rapid robust assay with high sensitivity. Under the established optimized condition, the concentration of gossypol giving 50% reduction of the maximum ELISA signal (I50) in the competitive standard curve was 0.20 microg/mL, whereas the detection limit for gossypol was 0.024 microg/mL. This ic-ELISA method for the analysis of gossypol extracted by methanol from a variety of cottonseed meals was further compared with the official method of the American Oil Chemists' Society (AOCS). The amounts of gossypol determined by the ic-ELISA had a good correlation with those obtained by the AOCS method (R2 = 0.90). 相似文献
16.
Monoclonal antibody-based fluorescence polarization immunoassay for sulfamethoxypyridazine and sulfachloropyridazine 总被引：1，自引：0，他引：1
Wang Z Zhang S Nesterenko IS Eremin SA Shen J 《Journal of agricultural and food chemistry》2007,55(17):6871-6878
In this paper, a new monoclonal antibody (Mab) against sulfamethoxypyridazine (SMP) was produced, and a fluorescence polarization immunoassay (FPIA) based on the produced Mab was developed and optimized for the qualitative screening analysis of SMP. The Mab was raised from mice immunized with SMP linked to bovine serum albumin (BSA) by carbodiimide activated ester formation, using a succinic anhydride spacer molecule between SMP and BSA. Fluorescein labeled sulfachloropyridazine (SCP) and SMP (tracer) were synthesized and purified by thin layer chromatography (TLC). The developed screening FPIA method can tolerate up to 20% methanol, and satisfactory assay sensitivity can be obtained between pH 4 and pH 8 and at lower salt concentration. The anti-SMP Mab exhibited a high cross-reactivity with SCP. The effect of the tracer structure on the analytical characteristic of the determination and on antigen-antibody binding constants was studied. The limits of detection (LOD) were 0.7 ng/mL for SMP and 0.25 ng/mL for SCP in buffer, respectively, whereas negligible cross-reactivities were exhibited by related sulfonamides. Analysis of SMP and SCP-fortified milk samples by the FPIA showed average recoveries from 60 to 145%. 相似文献
17.
Dang QL Kim WK Nguyen CM Choi YH Choi GJ Jang KS Park MS Lim CH Luu NH Kim JC 《Journal of agricultural and food chemistry》2011,59(20):11160-11167
The methanol extract of Annona squamosa seeds was highly active against two phytoparasitic nematodes, Bursaphelenchus xylophilus and Meloidogyne incognita. It efficiently suppressed plant diseases, caused by Phytophthora infestans and Puccinia recondita. Ten annonaceous acetogenins (AAs) were isolated, and their chemical structures were identified by mass and nuclear magnetic resonance spectral data. Out of 10 substances, eight displayed strong in vitro nematicidal activity against B. xylophilus with LD(50) values ranging 0.006 to 0.048 μg/mL. Squamocin-G showed potent nematicidal activity against M. incognita. Squamocin, squamocin-G, and squamostatin-A also displayed potent in vitro and in vivo antifungal activities against P. infestans causing tomato late blight. In addition, squamostatin-A effectively controlled the development of wheat leaf rust caused by P. recondita. Our findings suggested that A. squamosa seeds and its bioactive AAs can be an alternative resource of a promising botanical nematicide and fungicide to control various plant diseases. 相似文献
18.
Chen Y Wang Z Wang Z Tang S Zhu Y Xiao X 《Journal of agricultural and food chemistry》2008,56(9):2944-2952
A monoclonal antibody (Mab) was produced by using the kanamycin-glutaraldehyde-bovine serum albumin (Kan-GDA-BSA) conjugate as the immunogen. The anti-Kan Mab exhibited high cross-reactivity with tobramycin (Tob) and slight or negligible cross-reactivity with other aminoglycosides. The specificity and cross-reactivity of this Mab are discussed regarding the three-dimensional, computer-generated molecular models of the aminoglycosides. Using this Mab, a rapid enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based strip test for Kan and Tob were developed. The rapid ELISA showed a 50% inhibition value (IC 50) of 0.83 ng/mL for Kan and 0.89 ng/mL for Tob with the analysis time less than 40 min, and the recoveries from spiked swine tissues at levels of 25-200 microg/kg ranged from 52% to 96% for Kan and 61% to 86% for Tob. In contrast, the strip test for Kan or Tob had a visual detection limit of 5 ng/mL in PBS, 50 microg/kg in meat or liver, and 100 microg/kg in kidney, and the results can be judged within 5-10 min. Observed positive samples judged by the strip test can be further quantitated by ELISA, hence the two assays can complement each other for rapid detection of residual Kan and Tob in swine tissues. Moreover, physical-chemical factors that affected the ELISA and strip test performance were also investigated. The effect of pH and antibody amount for gold-antibody conjugation on the strip test sensitivity was determined followed by a theoretical explanation of the effects. 相似文献
19.
绿色高效杀线农药是现阶段防治植物线虫病的有效手段之一,针对在大规模杀线农药活性筛选测试阶段,传统人工镜检工作存在耗时长、准确率低、工作量大等问题,提出一种基于坐标注意力机制与高效边界框回归损失的线虫快速识别方法YOLOFN（YOLO for Nematodes）。基于YOLOv5s目标检测理论框架,在主干网络嵌入坐标注意力机制特征提取模块,融合线虫特征图位置信息于通道注意力中;进一步,平衡考量线虫目标的重叠比例、中心点距离、预测框宽高以及正负样本比例,以精确边界框回归的高效损失函数（Efficient IoU,EIoU）和焦点损失函数（Focal loss）优化定位损失函数和分类损失函数,最小化真实框与预测框的宽高差值,动态降低易区分样本的权重,快速聚焦有益训练样本,以提升模型对重叠黏连线虫目标的解析能力和回归精度。试验结果表明,YOLOFN在准确率、召回率和平均精度均值（mean Average Precision,mAP）性能指标上较改进前提高了0.2、4.4和3.8个百分点,与经典检测算法YOLOv3、SSD、Faster R-CNN3相比,mAP分别提高了1.1、31.7和15.1个百分点;与轻量化主干算法深度可分离卷积-YOLOv5、Mobilenetv2-YOLOv5、GhostNet-YOLOv5相比,在推理时间基本无差别情况下,mAP分别高出11、16.3和15个百分点。YOLOFN模型可快速、准确、高效完成线虫镜检统计工作,满足植物线虫病农药研发的实际需求,为加快植物线虫病防治新药的研制提供有力技术支持。 相似文献