草鱼呼肠孤病毒Ⅱ型VP6蛋白多克隆抗体制备及其特异性分析

为了制备高效的草鱼呼肠孤病毒Ⅱ型VP6蛋白多克隆抗体并对其特异性进行鉴定,实验以草鱼呼肠孤病毒Ⅱ型HZ08株为模板,采用PCR方法扩增S9基因,将该S9基因与p ET-32a(+)载体连接构建p ET-32a-S9原核表达载体,转化到大肠杆菌BL21(DE3)后用IPTG诱导表达;纯化后的重组VP6蛋白免疫新西兰兔,制备多克隆抗体,使用间接ELISA方法测定抗体效价,Western blot和IFA试验鉴定抗VP6蛋白多克隆抗体特异性。结果显示草鱼呼肠孤病毒Ⅱ型的S9基因在原核表达载体中能够正确地表达VP6蛋白,纯化的重组蛋白免疫新西兰兔制备的抗VP6多克隆抗体,经间接ELISA方法测定其效价约为1∶105,Western blot和IFA试验结果显示制备的多克隆抗体能特异性识别GCRVⅡ型毒株,而不能识别GCRV I型、Ⅲ型以及其它病毒,表明该多克隆抗体具有较高的特异性。研究表明制备的抗VP6蛋白多克隆抗体能够特异性识别GCRVⅡ型病毒,为GCRVⅡ型病原学研究及草鱼出血病临床诊断奠定基础。     

Cutaneous antibodies in excised skin from channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis

This study determined whether cutaneous antibodies were present in the excised skin of channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis Fouquet (Ich). Theronts were immobilized on or near the excised skin from immune fish. The survival of immobilized theronts was significantly reduced after exposure for 8 h to the culture of excised skin from immune fish. Culture fluids from excised skin of immune fish immobilized theronts with a peak in the immobilization titre at 24 h post-exposure. Immobility of theronts in the culture fluid from immune skin was removed after immunoabsorption with theronts. Indirect immunofluorescent staining of theronts treated with culture fluid from excised skin of immune fish revealed strong and uniform fluorescence on the cilia and cell surface of theronts. Western blot analysis of the culture fluid from immune fish revealed a 70-kDa band which corresponded to the molecular weight of catfish immunoglobulin heavy chain. The results of this study show that cutaneous antibodies to Ich theronts were present in and released from the excised skin from fish immune to Ich. Immobilization and killing of the theronts are two characteristics of the antibody response that appear to prevent the successful invasion of theronts into excised skin.     

Ⅱ型鲤疱疹病毒ORF121蛋白的多克隆抗体制备及鉴定

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。     

草鱼IFNa和IFNd重组蛋白表达、纯化及单克隆抗体制备

为系统研究草鱼I型干扰素的合成、分泌和免疫功能,本研究在大肠杆菌中表达并提纯了草鱼IFNa（CiIFNa）和IFNd（CiIFNd）重组蛋白。将CiIFNa和CiIFNd成熟肽分别克隆到pET-21d或pEHISTEVb表达载体上,并转化到大肠杆菌中;IPTG诱导表达得到CiIFNa和CiIFNd成熟肽的包涵体,经过盐酸胍变性、蛋白复性和浓缩后,利用AKTA分子筛层析获得了纯度较高的重组蛋白。用重组蛋白免疫小鼠,通过PEG法诱导得到杂交瘤细胞;将稳定分泌抗体的阳性细胞株的细胞悬液注射入小鼠腹腔,制备腹水抗体并进行纯化。本研究纯化了草鱼CiIFNa和CiIFNd各2株抗体,并采用SDS-PAGE、ELISA、Western blot和免疫荧光法对其进行了较全面的鉴定。研究结果表明CiIFNa和CiIFNd单克隆抗体特异性好、效价高,能够特异识别在大肠杆菌和真核细胞中表达的重组蛋白,且不存在CiIFNa和CiIFNd分子间的交叉识别。本研究制备的单克隆抗体为深入研究草鱼干扰素的细胞来源和蛋白表达规律奠定基础。     

草鱼IgM的表达及B淋巴细胞的抗菌活性

为了更加了解草鱼B淋巴细胞吞噬活性,本研究采用实时荧光定量PCR(qRTPCR)检测了IgM在胚胎发育中的表达量,并且检测了IgM在不同组织中的分布以及细菌刺激下IgM的转录情况。结果显示,IgM在卵裂期到出膜前期的表达量变化不明显,在出膜后开始显著增加;IgM在检测的组织中均有分布,在头肾中表达量最高,并且其表达量在不同细菌的刺激下均显著上调。从草鱼外周血中分离纯化得到B淋巴细胞,并通过吉姆萨染色、qRT-PCR和间接免疫荧光进行了验证。细菌吞噬实验结果显示,B淋巴细胞对嗜水气单胞菌具有一定的吞噬能力,并随孵育时间增加而逐渐增强。细菌刺激B淋巴细胞后活性氧(reactive oxygen species, ROS)和一氧化氮(nitric oxide,NO)释放量显著上升;血清调理实验结果显示,通过血清调理可以显著促进B淋巴细胞ROS的释放,然而对NO的释放水平没有显著影响。研究表明,草鱼B淋巴细胞对细菌具有一定的吞噬能力,并且可以通过呼吸爆发等非特异性免疫的方式直接参与抗菌免疫。     

Cutaneous antibodies from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis (Ich) may induce apoptosis of Ich theronts

This study explored the existence of apoptosis (programmed cell death) in Ichthyophthirius multifiliis Fouquet (Ich) theronts and determined the effect of cutaneous antibodies in skin culture fluid from fish immune to Ich on theront apoptosis. Apoptosis was detected in theronts and was clearly distinguished by fluorescent microscopy after staining with acridine orange and propidium iodide. The apoptotic theronts showed characteristic chromatin condensation and nuclear fragments containing chromatin pieces. The externalization of phosphatidylserine on the plasma membrane of apoptotic theronts was detected with fluorescein isothiocyanate-conjugated annexin using flow cytometry. Theront apoptosis was induced using the skin culture fluid from fish immune to Ich, which contained cutaneous antibodies against Ich. The highest apoptosis appeared in theronts exposed to immune skin culture fluid at a 1:10 dilution, compared with those at 1:20 and 1:40 dilutions. A direct correlation was noted between the percentage of apoptotic theronts and exposure duration to immune skin culture fluid. The study indicated that antibody reaction with theronts (immobilization) played an important role in theront apoptosis, but it could not be excluded that other components released from the excised skin had effects on theronts.     

草鱼干扰素3蛋白多克隆抗体制备及其应用

为探讨干扰素3(Interferon 3,IFN3)在抗病毒免疫应答中的作用,以草鱼(Ctenopharyngodon idella)巨噬细胞cDNA为模板,PCR扩增IFN3成熟肽基因序列,制备草鱼IFN3蛋白的多克隆抗体,同时研究了IFN3在草鱼不同免疫组织中的蛋白表达,以及草鱼呼肠孤病毒(grass carp reovirus,GCRV)感染草鱼肾细胞系(Ctenopharyngodon idella kidney,CIK)后不同时间点的表达。结果显示,细菌表达的重组IFN3大小约为45 kD,主要以包涵体形式存在;抗体效价约为1∶3 200,制备的多克隆抗体既能识别原核表达的重组蛋白,也能识别个体水平上和细胞水平上的内源蛋白。草鱼主要免疫组织中,肝胰腺和皮肤检测到相应条带。CIK细胞感染病毒后12 h开始检测到IFN3蛋白,随感染时间的延长,IFN3蛋白表达量有所增加。蛋白水平上检测IFN3的表达,为深入研究草鱼的抗病毒免疫机制奠定了基础。     

Antibody mediated immune response against Ichthyophthirius multifiliis using excised skin from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius

This study investigated antibody mediated immune response against Ichthyophthirius multifiliis (Ich) by determining whether theronts would retain the potential for reinfection, both in vitro and in vivo, after treatment with the culture fluid of excised skin from channel catfish, Ictalurus punctatus , immune to Ich. The invasion was reduced significantly ( P  < 0.05) for theronts treated with the immune culture fluid compared with those treated with the culture fluid from naive fish. The treatment of theronts with the immune culture fluid greatly reduced the size and survival of trophonts compared with those treated with the culture fluid from naive fish. Fewer fish were infected and the infection density was less for fish exposed to theronts treated with immune culture fluid. The infection was severe for fish invaded by theronts treated with the culture fluid from naive fish, with a high number of infected fish and heavy density of trophonts per fish. All fish were infected by Ich when exposed to the theronts treated with the immunoadsorbed culture fluid. In summary, results of this study show that cutaneous antibodies in the culture fluid of excised skin from immune fish significantly reduces theront infectivity by immobilizing or weakening theronts.     

Ⅱ型草鱼呼肠孤病毒VP4、VP35蛋白多克隆抗体制备及其免疫原性分析

为建立针对Ⅱ型草鱼呼肠孤病毒(GCRV)的血清学检测方法,分别构建了GCRV JX02株外衣壳蛋白VP4、VP35的原核重组表达质粒PGEX-4T-3-S6、PGEX-4T-3-S11,用纯化的重组蛋白r VP4、r VP35分别免疫小鼠制得相应的多克隆抗体,用间接ELISA方法测定2种抗体的效价,用Western Blot鉴定抗体的特异性。SDS-PAGE分析细菌表达的r VP4、r VP35大小分别约为98ku和61ku,且都主要以包涵体的形式存在;间接ELISA方法测定制备的抗体效价分别约为1:4×105和1:106;Western Blot结果显示,制备的2种多克隆抗体都既能够识别原核表达的重组蛋白,又能够识别JX02毒株上的对应蛋白,并且发现感染JX02的草鱼血清中存在结合VP4、VP35的相应抗体。本研究制备的2种多克隆抗体都具有良好的生物学特性,并且这2种重组蛋白作为相应抗体捕获原可以用于通过检测抗病毒抗体来确诊草鱼是否感染Ⅱ型GCRV。本研究将为GCRV主要流行株血清学检测方法的建立以及VP4、VP35蛋白相关功能研究奠定基础。     

Effect of fixation with formalin on flow cytometric measurement of DNA in nucleated blood cells

A study was performed to determine the role formalin fixation plays in flow cytometric (FCM) analysis of DNA in nucleated blood cells. Blood was frozen and stored at −80°C in a DMSO-citrate-sucrose buffer. When FCM analysis was needed the sample was thawed and added to a large volume of propidium iodide (PI) stain and analyzed after not less than 10 min of staining. No centrifugation steps were necessary. This protocol has been used by us for the discrimination of triploid from diploid Chinese grass carp (Ctenophary ngodon idella). Fixation of PI-staned nucleated RBCs with a final concentration of 1–2% phosphate-buffered formalin significantly lowered the amount of fluorescence recorded from the sample even when the fixed and stained RBCs were washed free of the fixative. Fixation with formalin prior to staining with PI yielded similar results. Our results show that formalin fixation of samples prior to or after staining with PI may lead to problems in data interpretation not previously appreciated. A FCM study was done to select the most appropriate biological DNA reference standard for grass carp RBCs. In this study DNA histograms of red blood cells from chicken, rainbow trout and diamondback watersnake as well as human nucleated white blood cells were compared to grass carp DNA histograms. Trout RBCs were selected as the most appropriate DNA reference standard for grass carp.     

草鱼肝细胞外泌体的分离鉴定及其对肝细胞miR-122/33和免疫相关基因表达的影响

外泌体是具有磷脂双分子膜结构的纳米级囊泡,能够参与机体多种生理过程。实验探讨了草鱼肝细胞外泌体的分离鉴定方法,并初步研究外泌体对草鱼肝细胞中miRNAs及免疫相关基因表达的影响。实验以草鱼肝细胞L8824为材料,通过超速离心获得外泌体,利用电子显微镜观察外泌体形态,采用纳米颗粒示踪分析(nanoparticle tracking analysis,NTA)技术检测外泌体粒径和数量,同时利用Western blot分析其标志蛋白CD63的表达,最后用正常肝细胞和油酸诱导的脂肪肝细胞源外泌体孵育草鱼肝细胞,通过Real-time qPCR技术检测两种不同来源的外泌体对草鱼肝细胞中miR-122/33及免疫相关基因(TNF-α,NF-κB,IL-1β,IL-6和IL-10)转录水平的影响。结果显示,草鱼肝细胞外泌体为30~150 nm的不均匀囊泡,呈圆形或椭圆形,有完整的膜结构;外泌体标志蛋白CD63呈阳性表达;NTA技术检测显示外泌体囊泡占所有囊泡的50%以上;脂肪肝细胞源外泌体显著提高了肝细胞中miR-122及炎症因子TNF-α、IL-1β和IL-6的mRNA转录水平。研究表明,通过超速离心法可成功分离草鱼肝细胞外泌体,且脂肪肝细胞源外泌体在草鱼肝细胞免疫调节中可能发挥重要作用。     

Characterization and application of monoclonal antibodies against white spot syndrome virus

Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG₁ class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies.     

Cloning and characterization of cDNA for carp matrix metalloproteinase 9

ABSTRACT: We have cloned a cDNA encoding the MMP-9 from a carp epidermal cell (EPC) cDNA library. The clone contains a 2025-base pair (bp) open reading frame encoding a protein of 674 amino acids. The deduced amino acid sequence shares 68% and 69% identity with medaka and Japanese flounder MMP-9. The hinge domain of the carp MMP-9, like those of the other non-mammalian species, lacks a type V collagen-like region that is typical of mammalian MMP-9. Gelatin zymography and immunoblot analysis of conditioned media of EPC cells and cDNA-transfected COS-7 cells detected a 76-kDa gelatinase. The apparent molecular mass of the carp zymogen is much smaller than those of its mammalian counterparts while almost identical with that of chicken 75-kDa gelatinase B-like enzyme. Although hypo-osmotic stress induced the elevation of MMP-9 mRNA level in EPC cells, no significant change in the protein in conditioned medium was detected during hypo-osmotic stress. Northern blot analysis detected a large amount of MMP-9 mRNA in carp kidney and spleen, suggesting the high expression of MMP-9 in blood cells, neutrophils, and macrophages. The smaller amount of MMP-9 mRNA was detected in gill, heart, fin, and eye, whereas none of the mRNA was detected in the hepatopancreas, intestine, brain, muscle, and skin.     

三疣梭子蟹颗粒血细胞单克隆抗体的研制及7种甲壳类动物血细胞的交叉反应研究

为研究虾蟹类甲壳动物血细胞膜表面是否具有相同的抗原决定簇及共同抗原表位的生物学特征,采用制备的抗三疣梭子蟹颗粒血细胞单克隆抗体,通过激光共聚焦扫描显微镜(laser scanning confocal microscopy,LSCM)、免疫印迹(Western blot)和流式细胞术(Flow Cytometry,FCM)等多种方法,测定了7种甲壳类动物(凡纳滨对虾、中国明对虾、刀额新对虾、口虾蛄、中华绒螯蟹、日本板蟹及美洲黄道蟹)颗粒血细胞及透明血细胞与三疣梭子蟹颗粒血细胞单克隆抗体发生特异性结合的抗原表位。LSCM可观察到该株抗三疣梭子蟹颗粒血细胞单克隆抗体与中华绒螯蟹和日本板蟹血细胞交叉反应结果为阳性,分析发现在中华绒螯蟹血淋巴中阳性颗粒血细胞在其颗粒血细胞总数中占76.74%,阳性透明血细胞在其透明血细胞总数中占70.59%,日本板蟹阳性颗粒血细胞在其颗粒血细胞总数中占73.86%,阳性透明血细胞在其透明血细胞总数中占16.67%,其余5种甲壳类动物均为阴性;Western blot测试结果显示该株单克隆抗体仅与中华绒螯蟹血细胞反应,且发生反应的抗原决定簇位于分子量为30 ku的蛋白带上;FCM分析发现该株单克隆抗体与中华绒螯蟹透明血细胞和颗粒血细胞均可发生交叉反应,阳性率分别为57.72%和77.05%,与日本板蟹透明血细胞阳性反应极少,阳性率仅为9.57%,与颗粒血细胞发生阳性反应的阳性率为82.59%。     

黑头软口鲦上皮瘤细胞ifn-1基因的表达及抗病毒活性

为了体外表达黑头软口鲦上皮瘤细胞(EPC)I型干扰素(IFN-1),本实验通过RTPCR从EPC中扩增ifn-1基因,构建重组表达质粒pET-32a-IFN-1,并转化到感受态细胞Transetta(DE3),体外纯化后检测其抗病毒活性。结果显示,ifn-1编码区大小为552 bp,编码184个氨基酸,与草鱼干扰素1(CiIFN1)亲缘关系最近。通过SDS-PAGE分析,重组表达质粒pET-32a-IFN-1在宿主菌中可明显表达约35 ku的融合蛋白条带,且部分呈可溶性表达,进而通过亲和纯化可溶性重组IFN-1(rIFN-1),免疫新西兰大白兔获得效价较高的抗IFN-1多克隆抗体,可用于检测细胞内源性的IFN-1。定量PCR显示rIFN-1与EPC细胞孵育可以诱导抗病毒蛋白Mx1的表达,并抑制鲤春病毒血症病毒(SVCV)引起的细胞病变(CPE)及SVCV的复制,表明rIFN-1具有抗病毒活性。     

中华鲟IFNe2的抗病毒天然免疫作用研究

为探究中华鲟干扰素e2（AsIFNe2）的免疫调控作用,本研究通过原核表达获得了重组中华鲟干扰素e2蛋白（rAsIFNe2）,并分析了它对抗病毒相关基因的影响及其抗病毒活性。实时荧光定量PCR（RT-qPCR）结果显示,rAsIFNe2能显著激活中华鲟鳍细胞中干扰素刺激基因（IFN-stimulated genes,ISGs）的表达,如Mx、PKR、Viperin和ADAR4 。此外,rAsIFNe2还能通过激活IRFs和IFNes基因的表达,从而帮助宿主细胞迅速建立抗病毒状态。在鲤春病毒血症病毒（spring viremia of carp virus,SVCV）感染鲤鱼上皮细胞（EPC）模型中,rAsIFNe2能够诱导EPC细胞中Mx、PKR和Viperin的表达,并降低EPC细胞中SVCV病毒G、N和P基因的表达,减少病变效应的产生。上述结果表明AsIFNe2在宿主抗病毒天然免疫反应中发挥作用,为深入了解中华鲟的干扰素免疫系统以及治疗病毒性疾病提供了理论依据。     

鱼类细胞的旋转管培养法

用圆柱形细胞培养瓶,对草鱼吻端组织ZC—7901细胞适应旋转培养条件进行了研究,测定了细胞接种浓度、旋转速度及细胞生长速度等有关参数。试验表明ZC—7901细胞能适应于旋转培养,适应后的细胞对草鱼出血病病毒仍具有敏感性。     

基因Ⅱ型草鱼呼肠孤病毒病毒样颗粒的构建与鉴定

为研究II型草鱼呼肠孤病毒(grass carp reovirus, GCRV)病毒样颗粒(viruses-like particles, VLPs)疫苗,本试验利用杆状病毒-昆虫细胞表达系统(baculovirus expression vector system, BEVS)将编码VP35蛋白的GCRV-S11基因克隆入杆状病毒载体pFastBacHTA^TM,然后将鉴定正确的重组质粒转化至DH10Bac感受态细胞,筛选得到重组穿梭质粒Bacmid-VP35。将穿梭质粒Bacmid-VP35以及实验室前期构建的重组穿梭质粒Bacmid-VP3、Bacmid-VP4分别转染sf9昆虫细胞获得重组杆状病毒pFHB-VP35、pFHB-VP3以及pFHB-VP4。利用Bac-PAK快速滴定试剂盒测定重组杆状病毒滴度,并通过间接免疫荧光(IFA)和Western Blot鉴定重组蛋白的表达情况。结果显示,本试验获得了较高滴度的重组杆状病毒,并且重组蛋白在杆状病毒感染的sf9昆虫细胞中正确表达。将成功表达的重组杆状病毒pFHB-VP35、pFHB-VP3以及pFHB-VP4共感染sf9细胞组装GCRV-VLPs,通过透射电镜(EM)进行检测。结果显示,GCRV的3个蛋白在sf9昆虫细胞中可以完成自我组装,形成与天然病毒结构形似的VLPs,直径大小为65-72nm。本试验结果为进一步研制安全、高效的GCRV-VLPs疫苗奠定基础。     

在草鱼免疫反应中莨菪佐剂性的初步观察

在草鱼免疫反应中莨菪佐剂性的初步观察赵建培（福州市郊区水产技术推广站，福州３５００１１）史维舟，陈昌福（华中农业大学水产学院，武昌４３００７０）已有研究报道指出，英若具有增强鱼类免疫力的作用［１］。但是，关于度著究竟是如何作用于鱼类的免疫系统，即为什...