鱼类病毒性出血性败血症病毒基质蛋白的原核表达及其亚细胞定位

为了对鱼类病毒性出血性败血症病毒(viral hemorrhagic septicemia virus, VHSV)基质蛋白(matrix protein, M)进行功能研究,本实验通过PCR扩增了M基因全长序列,将其克隆至原核表达载体pET-32a(+),转化至大肠杆菌Rosetta(DE3)感受态细胞后进行IPTG诱导表达,将纯化后的重组蛋白免疫BALB/c小鼠制备多克隆抗体,采用间接ELISA检测抗体效价,并运用Westernblot和间接免疫荧光检测抗体特异性。结果显示,M基因全长为606bp,IPTG诱导得到的融合蛋白主要以包涵体的形式存在,大小约为36 kD,比预计略小。间接ELISA检测抗体效价大于1:102400,Westernblot检测显示该抗体可以特异性识别纯化的融合蛋白和VHSV感染的鲤上皮瘤(epithelioma papulosum cyprini, EPC)细胞中的M蛋白。间接免疫荧光结果显示M蛋白多抗能识别感染VHSV的EPC细胞中的M蛋白,且M蛋白主要定位于细胞质和细胞膜。本研究中M蛋白多克隆抗体的制备将有助于开展M蛋白的功能研究及疾病的免疫学诊断。     

传染性造血器官坏死病毒抗原表位富集区的原核表达及应用

以传染性造血器官坏死病毒Sn1203株(IHNV-Sn1203)基因组RNA提取物为模板,利用生物信息学软件分析,通过RT-PCR一步法扩增截短的G蛋白基因序列(约375 bp),将其克隆到表达载体p ET-27b中,构建重组表达质粒p ET-27b-IHNV-short G,通过大肠杆菌Rosetta表达菌株获得高效表达。在IPTG浓度为0.25 mmol/L时,37℃诱导表达,经SDS-PAGE电泳分析显示目的蛋白相对分子质量约为14 000,符合预期大小,并以包涵体的形式表达,4 h时目的蛋白表达量最大。蛋白经变性、复性处理后获得不带任何标签的纯化蛋白,并利用该蛋白制备兔抗血清。ELISA结果显示,兔抗血清的效价为1∶80 000,说明制备的兔抗血清能够识别表达的重组蛋白;间接免疫荧光结果表明兔抗G蛋白血清具有良好的特异性,并且与VHSV参考毒株没有任何交叉反应。     

青鱼Dazl基因的原核表达及其多克隆抗体制备

实验进行了青鱼(Mylopharyngodon piceus)Dazl基因的原核表达,兔抗青鱼源Dazl多克隆抗体的制备及抗体的特异性验证。首先将青鱼Dazl基因的编码区利用重组表达引物从pCS2-MpDazl质粒中扩增出来,经酶切后连接到pET-28a载体中,构建pET-28a-MpDazl重组表达载体。然后将pET-28a-MpDazl重组质粒转化至大肠杆菌(Escherichia coli)BL21中,利用异丙基-β-d-硫代半乳糖苷(IPTG)诱导表达,获得分子量为27 kD的Dazl重组蛋白。将纯化的Dazl重组蛋白作为抗原免疫家兔制备多克隆抗体,抗体的效价和特异性通过ELISA法和Western blot检测。结果:成功构建重组表达载体pET-28a-MpDazl;以0.5 mmol/L IPTG在37℃条件下诱导4 h可获得高效表达的Dazl重组蛋白;制备的兔抗青鱼Dazl多克隆抗体能够特异性识别原核表达Dazl蛋白、青鱼卵巢的内源Dazl蛋白以及细胞中过表达的Dazl蛋白,并证实了青鱼Dazl蛋白在性腺中表达的特异性。     

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。     

黄鳝血清转铁蛋白多克隆抗体的制备及检测

为实现黄鳝血清转铁蛋白基因的原核表达并制备其多克隆抗体,利用基因特异性引物从黄鳝肝脏cDNA中扩增黄鳝转铁蛋白的C端序列,亚克隆至原核表达载体pET-28a(+)中,构建pET/Tf-C重组表达载体;转化大肠杆菌BL21(DE3)后进行IPTG诱导。利用Ni离子亲和层析技术纯化Tf-C蛋白,并免疫新西兰兔制备多克隆抗体;通过间接ELISA技术和组织蛋白印迹对制备的多克隆抗体进行检测。试验结果表明,成功构建pET/Tf-C原核表达载体,并实现了蛋白的表达和纯化;制备的多克隆抗体效价大于1∶25 600,并能特异性地识别来源于黄鳝不同组织的血清转铁蛋白。研究结果对黄鳝血清转铁蛋白功能的研究奠定了基础。     

团头鲂转录因子Nanog的原核表达及其多克隆抗体制备

为深入研究鱼类干细胞多能性转录因子Nanog的功能,本实验进行了团头鲂Nanog基因的原核表达和多克隆抗体的制备。首先,从团头鲂卵巢克隆出Nanog基因的编码区,将其连接到p ET32a载体上构建原核表达载体。接着将重组载体转化至大肠杆菌BL21(DE3)pLysS,通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,获得预期大小的Nanog重组蛋白,并获得大量蛋白以制备抗体。最后通过ELISA检测抗体的效价和采用Western blot技术检测Nanog抗体的特异性。结果显示,在37°C,0.5 mmol/L IPTG诱导4 h可获得Nanog重组蛋白的高效表达;制备的多克隆抗体能够有效识别原核表达的Nanog蛋白及团头鲂肝脏和精巢的内源Nanog蛋白;该抗体也可应用于检测HepG2细胞中过表达的团头鲂Nanog蛋白。本研究为蛋白的原核表达和特异性抗体的制备及验证提供了研究思路,也为研究鱼类Nanog基因功能提供了特异性抗体。     

鲤春病毒血症病毒核蛋白、磷蛋白与基质蛋白的表达、抗体制备及免疫原性比较

鲤春病毒血症病毒(SVCV)能够引起鲤科鱼类大量死亡,被世界动物卫生组织(OIE)列为必须申报的重要疫病,也是我国唯一被列为一类疫病的鱼类传染病。为建立SVCV的快速免疫学诊断方法,研究其主要结构蛋白间的免疫原性差异,实验首先采用原核表达系统克隆并诱导表达,纯化SVCV的核蛋白(N)、磷蛋白(P)和基质蛋白(M),并进一步免疫新西兰白兔制备抗血清,抗血清经Protein A柱进一步纯化获得3种蛋白的多克隆抗体。利用间接酶联免疫吸附测定(ELISA)和免疫印迹实验(Western blot)对抗体效价和特异性进行分析验证。结果发现,SVCV的N、P和M重组蛋白均在原核表达系统获得大量表达,且表达的蛋白经纯化后免疫实验动物产生了相应的多克隆抗体。Western blot结果显示,3种蛋白抗体均与SVCV重组蛋白及天然蛋白发生特异性的免疫反应。ELISA结果显示,针对P蛋白制备的抗体效价最高,可达409 600;针对N和M蛋白制备的抗体效价也均大于204 800。同时,特异性检测实验结果显示,制备的3种蛋白抗体均仅与SVCV发生特异性免疫反应,而与SVCV宿主其他易感病毒均不发生交叉反应。实验结果将对SVCV的快速诊断及疫苗开发提供新的手段和思路。     

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。     

鲤春病毒血症病毒G蛋白的原核表达及多克隆抗体的制备

利用PCR方法扩增鲤春病毒血症病毒糖蛋白G的部分基因序列,即全基因组序列上第3 094~4 170位的碱基,并将其克隆至表达载体pGEX-KG中,构建重组质粒SVCV-g-KG。将重组质粒转化感受态细胞BL21,经IPTG诱导后,表达了与预期大小相符的约66 kDa的融合蛋白,可溶性分析表明该蛋白主要表达在包涵体中。将纯化的融合蛋白免疫日本大耳白兔,制备多克隆抗体。酶联免疫吸附试验检测其抗体效价可达1∶256 000,间接免疫荧光试验和免疫印迹试验证明其与病毒结合活性良好,特异性高;病毒孵育试验结果表明该多克隆抗体具备中和活性,能够有效阻断鲤春病毒血症病毒对细胞的感染。     

草鱼呼肠孤病毒HZ08株S10编码蛋白多克隆抗体制备及其特性分析

为研究草鱼呼肠孤病毒(GCRV)HZ08株S10基因节段编码蛋白的可能功能,采用PCR方法扩增草鱼呼肠孤病毒HZ08株S10基因节段,并把该基因节段克隆至表达载体pET-32a(+),获得的重组表达载体pET-32a-S10转化到大肠杆菌BL21(DE3)菌株,用IPTG诱导表达,表达产物通过SDS-PAGE分析鉴定后,再通过变性、过Ni柱纯化、透析复性纯化获得目的蛋白。然后用纯化的重组蛋白免疫昆明小白鼠,制得多克隆抗体,用间接ELISA方法测定抗体效价,用Western blot和IFA(间接免疫荧光试验)鉴定抗体特异性。结果表明,SDS-PAGE分析表达的重组蛋白约为53 ku,大小与预期相符,目的蛋白主要存在于包涵体中;过Ni柱纯化、透析复性纯化后的重组蛋白纯度可达97.4%;间接ELISA测得制备的多克隆抗体效价约为1∶106,Western blot和IFA结果显示,制备的多克隆抗体能识别HZ08毒株,表明S10编码蛋白为GCRV-HZ08株的结构蛋白。     

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land‐based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real‐time RT‐PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re‐isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%–50% of the cohabiting lumpfish survived after 4 weeks. 80%–92% of the Atlantic salmon survived, but no viral RNA was detected by real‐time RT‐PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.     

A survey of wild marine fish identifies a potential origin of an outbreak of viral haemorrhagic septicaemia in wrasse,Labridae, used as cleaner fish on marine Atlantic salmon,Salmo salar L., farms

Viral haemorrhagic septicaemia virus (VHSV) was isolated from five species of wrasse (Labridae) used as biological controls for parasitic sea lice predominantly, Lepeophtheirus salmonis (Krøyer, 1837), on marine Atlantic salmon, Salmo salar L., farms in Shetland. As part of the epidemiological investigation, 1400 wild marine fish were caught and screened in pools of 10 for VHSV using virus isolation. Eleven pools (8%) were confirmed VHSV positive from: grey gurnard, Eutrigla gurnardus L.; Atlantic herring, Clupea harengus L.; Norway pout, Trisopterus esmarkii (Nilsson); plaice, Pleuronectes platessa L.; sprat, Sprattus sprattus L. and whiting, Merlangius merlangus L. The isolation of VHSV from grey gurnard is the first documented report in this species. Nucleic acid sequencing of the partial nucleocapsid (N) and glycoprotein (G) genes was carried out for viral characterization. Sequence analysis confirmed that all wild isolates were genotype III the same as the wrasse and there was a close genetic similarity between the isolates from wild fish and wrasse on the farms. Infection from these local wild marine fish is the most likely source of VHSV isolated from wrasse on the fish farms.     

A combined phage display ScFv library against Myxobolus rotundus infecting crucian carp, Carassius auratus auratus (L.), in China

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%.     

Differential viral haemorrhagic septicaemia virus genotype IVb infection in fin fibroblast and epithelial cell lines from walleye,Sander vitreus (Mitchill), at cold temperatures

A cell line, WE‐cfin11e, with an epithelial‐like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast‐like cell line, WE‐cfin11f, and compared with WE‐cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens‐1 (ZO‐1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE‐cfin11e stained for ZO‐1 and only WE‐cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE‐cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE‐cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE‐cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism.     

Epidemiological aspects of viral haemorrhagic septicaemia virus genotype II isolated from Baltic herring, Clupea harengus membras L

This study was carried out to clarify the role of wild fish, especially Baltic herring, Clupea harengus membras L., in the epidemiology of viral haemorrhagic septicaemia virus (VHSV) in brackish water in Finland. Baltic herring with no visible signs of disease were collected from the Archipelago Sea, the Gulf of Bothnia and the eastern Gulf of Finland. In total, 7580 herring were examined by virus isolation as 758 pooled samples and 3029 wild salmonid broodfish as pooled samples during 2004-2006. VHSV was isolated from 51 pooled herring samples in bluegill fibroblast-2 cells, but not in epithelioma papulosum cyprini cells. The majority of isolations were from the coastal archipelago and from fish caught during the spawning season. Based on glycoprotein (G) gene sequences, the virus was classified as a member of genotype II of VHSV. Pairwise comparisons of the G gene regions of herring isolates revealed that all the isolates were closely related, with 98.8-100% nucleotide homology. Phylogenetic analyses revealed that they were closely related to the strains isolated previously from herring and sprat, Sprattus sprattus (L.), in Gotland and to the VHSV isolates from European river lamprey, Lampetra fluviatilis (L.), in the rivers that flow into the Bothnian Bay. The infection in Baltic herring is likely to be independent of the VHSV Id epidemic in farmed rainbow trout, Oncorhynchus mykiss (Walbaum).     

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti‐rVP28 antiserum to detect the presence of the virus in WSSV‐infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR‐positive WSSV‐infected shrimp samples. Immunodot blot was found to be equivalent to first‐step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 10⁵ viral DNA copies.     

Change of viral hemorrhagic septicemia virus (VHSV) titer in olive flounder (Paralichthys olivaceus) following Poly(I:C) administration

Olive flounder (Paralichthys olivaceus) are highly protected from a viral hemorrhagic septicemia virus (VHSV) challenge following Polyinosinic–polycytidylic acid [Poly(I:C)] administration. In the present study, we investigated the change of VHSV titer in olive flounder following Poly(I:C) administration to understand virus dynamics in the fish. Fish challenged with VHSV that were not administered Poly(I:C) showed 63.8 % cumulative mortality. VHSV was detectable the next day after VHSV challenge and multiplied very quickly to around 10^7.5 TCID₅₀/g in 3 days. About 10⁷ TCID₅₀/g titer was maintained until 7 days and then subsequently decreased and almost disappeared after 21 days. In contrast, 1.7 % cumulative mortality was observed in fish administered Poly(I:C), and no VHSV titer was detected in almost all fish for 28 days. These results confirm that multiplication of VHSV is strongly down-regulated in olive flounder following Poly(I:C) administration.