传染性造血器官坏死病病毒参考蛋白的研制

为规避活病毒作参考物质存在的生物安全风险,本研究利用毕赤酵母表达系统表达了传染性造血器官坏死病病毒(IHNV)的糖蛋白来制备参考蛋白,作为IHNV免疫学检测参考物质。本研究选用IHNV糖蛋白基因(IHNV-G)为目的基因,根据Gen Bank中IHNV全基因序列设计特异性引物,以IHNV-uk株病毒核酸为模板,通过RT-PCR获得糖蛋白基因片段,将其克隆至真核表达载体p PICZαA,转入毕赤酵母感受态细胞GS115中,使外源基因与酵母基因融合,通过1%甲醇诱导表达外源蛋白,SDS-PAGE分析显示获得可溶性表达蛋白,分子量大于70 ku。经Western Blot和ELISA分析,该表达产物可以被IHNV多抗特异性识别。0.1531 mg的重组蛋白与0.3125TCID50 IHNV在ELISA实验中反应原性相当,–20°C可稳定保存2个月,说明其在一定程度上可替代病毒作为参考物质。该研究为IHNV ELISA检测试剂盒的开发奠定基础。     

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。     

鲤春病毒血症病毒糖蛋白的截短表达及其免疫原性分析

为了揭示鲤春病毒血症病毒(spring viremia of carp virus,SVCV)糖蛋白编码基因的主要免疫原性决定区域,本研究对SVCV的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar 6.0软件对SVCV糖蛋白基因的跨膜区、亲水性以及抗原表位进行分析后,采用RT-PCR对该基因主要抗原决定区域编码片段进行扩增,并构建重组表达质粒p GEX-Gtr,对其进行诱导表达后获得截短的SVCV糖蛋白的重组蛋白。将表达的重组蛋白进行纯化复性后,作为免疫原制备兔抗血清,采用ELISA法检测其效价,采用免疫印迹以及间接免疫荧光技术检测该重组蛋白的免疫原性。研究结果显示,截短表达的糖蛋白编码基因长1317 bp,编码439个氨基酸(29~467),推测分子量为49.6 k D。利用该重组蛋白制备的兔抗血清,其与重组蛋白的反应效价为1∶64000,。免疫印迹及间接免疫荧光结果显示,该兔抗血清与SVCV-HN株能发生特异性反应,表明该重组蛋白与天然的病毒表面糖蛋白的免疫原性无差异。上述研究结果表明,截短表达的重组蛋白具有很好的免疫原性,可应用于免疫诊断技术研发与基因工程疫苗的研制。     

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VP4蛋白单克隆抗体的制备及鉴定

为了建立针对草鱼呼肠孤病毒(GCRV)流行株的血清学检测方法,实验构建了能高效表达GCRV HZ08株主要衣壳蛋白VP4的重组表达载体pET32a-S6,利用纯化的VP4重组蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/O细胞融合,经过克隆和筛选,获得3株能稳定分泌抗VP4重组蛋白单克隆抗体(monoclonal antibody,MAb)的杂交瘤细胞,分别命名为2C2、2F3和5E5.抗体经亚型鉴定均为IgG1,轻链为K链;间接ELISA试验证明,3株杂交瘤细胞分泌的MAb可特异性识别GCRV-HZ08,与GSRV,ISKNV,IHNV均无交叉反应.选择2C2作为腹水生产细胞株,免疫小鼠后腹水ELISA效价为1∶720 000;IFA和Western-blotting结果显示,这株杂交瘤细胞分泌的MAb能够特异性识别GCRV-HZ08病毒粒子.本实验制备的MAb具有良好的生物学特性,为GCRV流行株血清学检测方法的建立及VP4蛋白相关功能研究奠定了基础.     

鳜传染性脾肾坏死病毒主衣壳蛋白单克隆抗体的制备及鉴定

为了建立鳜传染性脾肾坏死病毒(ISKNV)疫苗抗原含量的ELISA检测方法,制备了3株抗ISKNV主衣壳蛋白(MCP)的单克隆抗体,鉴定了其生物学特性。将大肠杆菌表达的重组MCP纯化复性后,连续3次免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与SP2/0细胞融合,经过克隆、筛选,获得3株能稳定分泌抗ISKNV MCP蛋白的单克隆抗体阳性细胞株,分别命名为5F1、3D9和5B4,均为Ig G1亚型。间接ELISA实验表明,3株单抗可特异性识别ISKNV,与鳜弹状病毒、大鲵虹彩病毒等无交叉反应。将5F1株免疫小鼠后制备腹水,以重组MCP和ISKNV细胞培养物上清液为检测抗原,ELISA检测腹水效价分别为1∶51 200和1∶400。间接免疫荧光(IFA)和Western Blotting鉴定结果显示,5F1能够与ISKNV病毒发生特异性反应,并初步确定5F1单抗株制备的腹水用于IFA的使用浓度为1∶200、Western Blotting的使用浓度为1∶1000。结果证实,成功制备了抗ISKNV MCP的单克隆抗体,可特异性识别ISKNV病毒粒子和MCP蛋白,为建立ISKNV疫苗抗原含量检测方法奠定了基础。     

鲑鱼甲病毒E1基因高保守区融合表达及免疫特性的分析

根据Gen Bank中公布的鲑鱼甲病毒(salmonid alphavirus,SAV)SAV 1、SAV 2和SAV3三个基因型中E1基因,选择高保守序列702 bp(436-1137)合成基因,命名为SAV E1,将其克隆到原核表达载体p Cold TF中构建重组质粒。然后将重组质粒转化到大肠杆菌感受态细胞BL21中,经终浓度为1.0 mmol/L的IPTG诱导表达,SDSPAGE和Western blot鉴定,重组蛋白均获得了表达,表达E1重组蛋白约95 k D。用镍离子亲和层析柱纯化重组蛋白,制备抗血清。间接ELISA结果显示,鼠抗重组蛋白E1血清效价为1∶25 600;间接免疫荧光结果显示,鼠抗重组E1蛋白血清可与SAV发生特异反应,由此表明表达的E1重组蛋白具有良好的免疫原性和免疫反应性,为SAV检测方法的建立提供理论依据。     

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。     

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。     

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。     

Inactivated infectious haematopoietic necrosis virus (IHNV) vaccines

The inactivation dynamics of infectious haematopoietic necrosis virus (IHNV) by b-propiolactone (BPL), binary ethylenimine (BEI), formaldehyde or heat and the antigenic and immunogenic properties of the inactivated vaccines were evaluated. Chemical treatment of IHNV with 2.7 mm BPL, 1.5 mm BEI or 50 mm formaldehyde abolished virus infectivity within 48 h whereas heat treatment at 50 or 100 degrees C rendered the virus innocuous within 30 min. The inactivated IHNV vaccines were recognized by rainbow trout, Oncorhynchus mykiss, IHNV-specific antibodies and were differentially recognized by antigenic site I or antigenic site II IHNV glycoprotein-specific neutralizing monoclonal antibodies. The BPL inactivated whole virus vaccine was highly efficacious in vaccinated rainbow trout challenged by waterborne exposure to IHNV 7, 28, 42 or 56 days (15 degrees C) after immunization. The formaldehyde inactivated whole virus vaccine was efficacious 7 or 11 days after vaccination of rainbow trout but performed inconsistently when tested at later time points. The other vaccines tested were not efficacious.     

Real-time PCR for the detection and quantitative analysis of IHNV in salmonids

The rapid identification and quantification of virus in diseased fish is a goal both conservationists and commercial aquaculturists have struggled to attain. Recently a technique for the detection of viral mRNA particles that uses fluorescent tagging and amplification has been developed. Utilizing primers and fluorescent labelled probes generated for the specific identification of the nucleocapsid (N) and glycoprotein (G) genes of infectious haematopoietic necrosis virus (IHNV), and an instrument that measures cyclic emittance of fluorescence, the presence or absence of virus can be easily and rapidly confirmed. This method is not only useful in confirming viral presence but is effective in measuring the relative or absolute quantity of virus present within the sample. This allows for the determination of the health status of a carrier fish by measuring the quantity of viral genomes or transcribed viral genes present. Because this method is based on sequence detection, instead of virus isolation in cell culture, it is also effective in determining the presence of pathogenic organisms from water, fish feeds, or other potential reservoirs of infection.     

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put‐and‐take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put‐and‐take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co‐infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV‐positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co‐infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus‐introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.     

An isolate and sequence database of infectious haematopoietic necrosis virus (IHNV)

In the field of fish diseases, the amount of relevant information available is enormous. Internet‐based databases are an excellent tool for keeping track of the available knowledge in the field. Fishpathogens.eu was launched in June 2009 with the aim of collecting, storing and sorting data on fish pathogens. The first pathogen to be included was the rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Here, we present an extension of the database to also include infectious haematopoietic necrosis virus (IHNV). The database is developed, maintained and managed by the European Community Reference Laboratory for Fish Diseases and collaborators. It is available at http://www.fishpathogens.eu/ihnv .     

两种检测传染性造血器官坏死病(IHNV)方法的比较

传染性造血器官坏死病(IHNV)是一种危害极其严重的鱼类疾病。本实验用一步RT-PCR法和套式PCR法对IHNV的基因进行扩增,通过扩增片段的琼脂糖电泳分析,来判定病鱼是否携带IHNV。实验结果表明,通过一步法RT-PCR可扩增出IHNV的693bp片段;通过套式PCR法可扩增出IHNV的786bp片段和323bp片段。两种方法均能够检测待测样品中含有IHNV。比较发现一步RT-PCR法比套式PCR法更加快速、简便、敏感,减少了被污染机会。     

Characterization of susceptibility and carrier status of burbot,Lota lota (L.), to IHNV,IPNV, Flavobacterium psychrophilum,Aeromonas salmonicida and Renibacterium salmoninarum

In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.