鲤疱疹病毒Ⅱ型ORF66基因DNA疫苗载体的构建及其免疫效果

鲤疱疹病毒Ⅱ型(CyHV-2)能够引起鲫大量死亡,严重威胁我国水产养殖业的健康发展,目前,针对CyHV-2尚无有效的商业化疫苗或治疗措施。为构建CyHV-2DNA疫苗,本研究将其衣壳蛋白ORF66编码基因克隆至pVAX1真核表达载体上,构建重组质粒pVAX-ORF66。此外,在BL21(DE3)pLysS中对ORF66蛋白进行了原核表达,表达蛋白经纯化后免疫新西兰白兔制备了ORF66特异性抗体。酶联免疫吸附(ELISA)检测显示,制备抗体效价达1∶20000以上。将pVAX-ORF66质粒转染金鱼脑细胞系(GFB),利用制备的ORF66抗体进行间接免疫荧光(IFA)检测,结果显示,ORF66蛋白可以在细胞中大量表达,且主要定位于细胞质中。将pVAX-ORF66质粒肌肉注射鲫后进行CyHV-2免疫保护实验,结果表明,其相对免疫保护率达55.6%。本研究针对CyHV-2构建了一种制备简单、成本低廉的DNA疫苗,为鲫造血器官坏死病的免疫预防及感染分子机制研究奠定了前期实验基础。     

DNA疫苗的研究进展

为开发针对鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3) [ 又称为锦鲤疱疹病毒(Koi herpesvirus,KHV)] 的DNA疫苗,该研究将CyHV-3 ORF65插入pEGFP-N1,构建pEGFP-ORF65重组质粒,通过转染实验与活体成像实验证实pORF65-EGFP融合蛋白可以在建鲤(Cyprinus carpio var. Jian)体外、体内表达;重组质粒按照3 μg·尾⁻¹的剂量尾柄肌肉注射免疫10 g左右的建鲤鱼苗,免疫3次,ELISA检测表明pEGFP-ORF65免疫后可以显著提高血清特异性抗体水平;攻毒实验显示,CyHV-3攻毒21 d后,PBS组、pEGFP-N1组和pEGFP-ORF65组的建鲤成活率分别为25%、30%和90%,攻毒建鲤的成活率显著提高(P<0.01)。该研究为ORF65作为DNA疫苗靶基因应用于CyHV-3的防控提供了依据。

     

鲤疱疹病毒3型ORF136基因编码蛋白多克隆抗体的制备与鉴定

为了对鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)ORF136基因编码蛋白进行功能研究和血清学诊断,本实验通过对ORF136基因推导的第31~157位氨基酸序列进行PCR扩增,并与原核载体pET-32a(+)连接,转化至大肠杆菌Rosetta(DE3)感受态后进行IPTG诱导表达,将纯化后的重组蛋白免疫新西兰白兔(Oryctolagus cuniculus)以制备ORF136多克隆抗体,运用Western blot和间接免疫荧光技术对抗体进行鉴定。结果表明,重组融合表达蛋白大小与预期一致,约为35 kD,且主要分布在包涵体中。Western blot分析显示,免疫兔后获得的纯化ORF136多克隆抗体能特异性识别纯化的CyHV-3和感染CyHV-3的KS细胞;间接免疫荧光分析进一步表明ORF136多抗能识别感染CyHV-3的KS细胞。ORF136多克隆抗体的制备为ORF136蛋白功能研究和CyHV-3血清学诊断方法的建立提供了重要基础。     

鲤疱疹病毒Ⅱ型ORF4基因的克隆、表达与免疫学检测方法

根据鲤疱疹病毒II型(Cyprinid herpesvirus Ⅱ,Cy HV-2)ORF 4基因序列(Gen Bank:JQ815364.1)设计特异性引物,PCR扩增得到ORF 4基因编码框全长序列1 041 bp,将其克隆至原核表达载体p ET-32a(+)中,构建了重组原核表达载体p ET-32a-ORF 4。将p ET-32a-ORF 4重组载体转化大肠杆菌BL21(DE3),经IPTG诱导得到融合表达的重组蛋白,融合表达的重组蛋白主要以包涵体的形式存在,其分子质量约为57 ku,与预期大小一致。将纯化的重组蛋白免疫日本大耳兔,制备了多克隆抗体,ELISA检测抗体效价大于1∶50 000,Western blot检测显示该抗体可以特异性识别重组蛋白。间接免疫荧光检测结果表明:该多克隆抗体可与由Cy HV-2感染引起细胞病变的异育银鲫脑组织细胞(GICB)发生特异性的结合。     

鲤疱疹病毒3型T分离株主要免疫原性蛋白的鉴定

为了鉴定鲤疱疹病毒3型(Cyprinid Herpesvirus 3,CyHV-3)主要免疫原性蛋白,研究采用CyHV-3-T分离株感染CCB细胞系,运用蔗糖密度梯度超速离心方法对CyHV-3进行纯化,纯化的病毒颗粒经SDSPAGE和考马斯亮蓝染色后,用锦鲤(Cyprinus carpio haematopterus)抗CyHV-3阳性血清进行Western blotting分析和液相色谱串联质谱鉴定。结果表明,透射电镜下可观察到大量完整囊膜包裹或只有裸露核衣壳的CyHV-3颗粒,Western blotting结果显示抗CyHV-3阳性血清与多种病毒蛋白具有明显特异性免疫反应,质谱鉴定表明其中4种免疫原性蛋白分别为ORF92、ORF66、ORF72和ORF81,其中ORF66和ORF72为首次鉴定的具有免疫原性衣壳蛋白。本研究将为CyHV-3血清学诊断方法的建立、亚单位疫苗或DNA疫苗的研制提供更多候选抗原。     

锦鲤疱疹病毒3型ORF126基因克隆及生物信息学分析

为研究锦鲤疱疹病毒3型(KHV-3)ORF126基因编码蛋白的功能,我们对该蛋白的结构特征进行研究分析。本实验采用PCR扩增技术获得KHV ORF126基因的完整序列,构建pMD19-T-ORF 126重组质粒,应用生物信息学方法分析ORF126基因编码蛋白的理化特征。结果显示:KHV ORF126基因翻译合成273个氨基酸;ExPasy预测该蛋白的理论分子量为29.9kDa,等电点为5.11;信号肽的切割部位最可能位于第19~20位氨基酸之间;跨膜区位于第145~168位氨基酸;抗原表位预测显示抗原性良好;编码蛋白不含N-糖基化位点,含有4个O-糖基化位点和17个磷酸化位点。     

鲤疱疹病毒Ⅱ型主要免疫原性蛋白的鉴定

为了鉴定CyHV-2的主要免疫原性蛋白,本研究用分离的CyHV-2-YC-1分离株(下简称CyHV-2)感染异育银鲫尾鳍细胞系(GiCF),用蔗糖密度梯度超速离心法对细胞感染液中的CyHV-2进行纯化,纯化后的CyHV-2病毒免疫小鼠制备抗CyHV-2多克隆抗体。纯化的病毒颗粒经变性聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝染色后,用抗CyHV-2多克隆抗体进行Western blotting分析和质谱鉴定。结果显示,透射电镜下观察发现50%～66%的蔗糖梯度多见完整囊膜包裹的CyHV-2病毒颗粒,也有少量囊膜破损的病毒颗粒。Western blotting结果显示,抗CyHV-2多克隆抗体与多种病毒蛋白具有特异性免疫反应,质谱鉴定显示,其中8种主要免疫原性蛋白分别是ORF92、ORF115、ORF25、ORF57、ORF66、ORF72、ORF131和ORF132。研究表明,通过蔗糖密度梯度超速离心法提纯CyHV-2病毒颗粒后,本研究制备的抗CyHV-2多克隆抗体能够特异性识别CyHV-2病毒的主要免疫原性蛋白。本研究将为CyHV-2免疫学检测方法的建立以及疫苗的研制提供更多...     

一种基于Ⅱ型鲤疱疹病毒衣壳蛋白72的免疫学检测方法

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。     

鲤疱疹病毒Ⅱ型对异育银鲫背鳍细胞的显微形态与免疫基因表达水平的影响

为研究鲤疱疹病毒Ⅱ型(Cy HV-2)体外感染复制特征以及异育银鲫抗病毒免疫应答反应。本实验采用组织块培养法建立了异育银鲫背鳍细胞的原代培养体系。结果显示,在10 d左右可观察到组织块迁移分离出新的单层细胞,3周左右细胞可覆盖底部面积为25cm2培养瓶的底部;经Cy HV-2悬液感染离体培养的原代细胞,3 d后病毒滴度增殖至106拷贝/m L;在病毒感染6 d后出现典型的细胞病变效应;Cy HV-2感染原代细胞后,分析前期通过鱼体水平实验鉴定出的与该病毒感染相关的免疫基因:PNP5a、MPO、MHCⅠ、LYZ-C、IL-11、ITLN、PNP5a和DUSP,Real-time Rt-PCR结果显示大部分基因在细胞水平均有显著性的上调,与鱼体水平实验结果一致。本研究建立了原代培养的异育银鲫背鳍细胞,用于构建体外感染Cy HV-2病毒的细胞模型,为深入研究Cy HV-2的感染复制规律及其与宿主的相互作用关系,以及细胞水平筛选抗病毒药物实验奠定了基础。     

Efficacy of koi herpesvirus DNA vaccine administration by immersion method on Cyprinus carpio field scale culture

Koi herpesvirus specifically infects and causes mass mortality on koi and carp, resulting in severe economic losses. In this study, we presented the efficacy of KHV DNA vaccine administration by immersion method on Cyprinus carpio. Two different immersion densities of fish were applied, namely 800 fish L^?1 and 1200 fish L^?1. Thirty‐day‐old common carp juveniles were immersed for 30 min in the water containing 1.3 × 10⁸ CFU mL^?1 of heat‐killed Escherichia coli carrying DNA vaccine encoding glycoprotein‐25, and without vaccination treatment as controls. The challenge test was performed at 30 days post vaccination by injecting 0.1 mL KHV filtrate (10^?3 of dilution rate). The result showed that higher relative per cent survival of KHV‐challenged fish was obtained in 800 fish L^?1 (P < 0.05). Furthermore, significant specific antibody anti‐KHV response (P < 0.05) was detected on 28 and 36 days post vaccination in 800 and 1200 fish L^?1, respectively, compared to the controls there was no specific antibody detected. In conclusion, the KHV DNA vaccine could provide good protection in common carp against KHV infection, which has practical applications in aquaculture practices.     

Antiviral activities of Clinacanthus nutans (Burm.f.) Lindau extract against Cyprinid herpesvirus 3 in koi (Cyprinus carpio koi)

Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC₅₀) of extract was higher than 5 mg/ml. The 50% effective dose (ED₅₀) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED₅₀ from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.     

锦鲤疱疹病毒GZ1301株的分离与鉴定

鲤疱疹病毒3型(cyprinid herpesvirus 3,CyHV-3)囊膜蛋白ORF65基因全长1 785 bp,编码594个氨基酸。该研究在稀有密码子分析及信号肽与跨膜结构预测的基础上,将pORF65中的N端信号肽与C端跨膜区段删除后进行密码子优化与基因合成,将合成的截短 ORF65 (truncated ORF65) 插入pET32a (+)载体,构建了pET32a-trunORF65;进一步采用DNAstar、ABCpred、BepiPred 1.0软件预测了pORF65的5个B细胞表位优势区段,以合成的截短ORF65为模板,通过SOE-PCR将5个B细胞表位优势区段编码序列融合后插入pET32a (+)载体,构建了pET32a-modORF65。重组质粒分别转入BL21 (DE3)菌株,经IPTG诱导,SDS-PAGE和Western-blot分析,pET32a-modORF65获得高效表达,表达的融合蛋白分子量为56.4 kD。此外,利用rProtein G亲和层析纯化了锦鲤(Cyprinus carpio haematopterus)血清IgM,免疫小鼠,制备了鼠抗锦鲤IgM多克隆抗体。在上述研究的基础上,将纯化的pORF65作为包被抗原,鼠抗锦鲤IgM多克隆抗体作为检测抗体,建立了间接ELISA方法,该方法可以检测pEGFP-ORF65 DNA疫苗免疫锦鲤后产生的特异性抗体。     

Construction of KHV‐CJ ORF25 DNA vaccine and immune challenge test

KHV ORF25 fragments were cloned from Koi Herpes Virus‐CJ (KHV‐CJ) strains isolated in our laboratory. The amplified products were inserted into the eukaryotic expression vector pIRES‐neo, forming recombinant plasmid pIRES‐ORF25. The recombinant plasmid pIRES‐ORF25 at 1 μg per koi, 10 μg per koi and 50 μg per koi was intramuscularly injected into healthy kois, respectively. The results showed that the recombinant pIRES‐ORF25 could induce the production of specific antibodies in koi determined by indirect ELISA. The differences of immune effect between three doses were not significant (P > 0.05), but all of them could induce the production of neutralizing antibodies. The immune challenge test showed that the mortality of koi injected with PBS, blank pIRES‐neo vector and nothing was 90%, 92.5% and 85% at 25 days. While the mortalities of koi injected with eukaryotic expression plasmid pIRES‐ORF25 were 20%, 17.5% and 12.5%. Differences in comparison with the control group were highly significant (P < 0.01). Histopathological staining revealed that the tissues of the immunized koi did not change apparently. In conclusion, the DNA vaccine pIRES‐ORF25 construct could well protect koi against KHV and had the potential to be applied in practice.     

建鲤基因组中一个ty3-gypsy反转录转座子的发现与分析

转座子是动植物基因组的重要组成部分,在前期研究中发现建鲤(Cyprinus carpio var.jian)基因组中存在一个ty3-gypsy反转录转座子类型的转座子,并将其命名为JRE转座子(Jian carp Retrotransposon,JRE)。为了研究JRE反转录转座子在建鲤基因组中的功能,采用PCR扩增、荧光定量PCR和原位杂交等方法对JRE转座子的特性进行了研究。JRE反转录转座子全长5126 bp,具有5?端470 bp和3?端453 bp长末端重复片段(long terminal repeat end,LTR),中间的开放阅读框(ORF)包括核心蛋白基因(gag)和酶基因区域(pol),其长度为4203 bp。pol基因具有典型的ty3-gypsy反转录转座子结构,基因顺序为PR-RT-RH-IN基因。对pol基因的同源分析表明,其与虾夷扇贝(Mizuhopecten yessoensis)、栉孔扇贝(Azumapecten farreri)、大堡礁海绵(Xiphophorus maculates)和斑剑尾鱼(Xiphophorus maculates)pol基因相似性分别为40.7%、40.0%、32.8%和30.1%,因此JRE可能属于JULE反转录转座子家族。采用实时定量PCR对JRE转座子在建鲤基因组内的拷贝数进行了测定,结果表明其拷贝数为124,同时对不同组织中的m RNA表达量的研究表明,JRE转座子在建鲤肝、肾、血、肌肉、性腺5种组织中均有表达,在肾和肝中表达量略高。染色体原位杂交结果表明,JRE转座子在建鲤的染色体上随机分布,没有明显的规律性。本研究表明,JRE转座子具有典型的反转录转座子结构,属于JULE转座子的分枝,在染色体上的分布不多,其转录活性并不是很高,对我们了解建鲤基因组构成和特点增加了知识储备,同时为利用转座子的活性进行转基因研究提供了一种新的途径和工具。     

Validation of a serum neutralization test for detection of antibodies specific to cyprinid herpesvirus 3 in infected common and koi carp (Cyprinus carpio)

Cyprinid herpesvirus 3 (CyHV‐3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV‐3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV‐3‐specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV‐3‐infected carp. French CyHV‐3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV‐3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post‐infection. The results suggest that this non‐lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV‐3 disease.     

Cyprinid herpesvirus 3 as a potential biological control agent for carp (Cyprinus carpio) in Australia: susceptibility of non‐target species

Carp (Cyprinus carpio L.) is a pest species in Australian waterways, and cyprinid herpesvirus 3 (CyHV‐3) is being considered as a potential biological control (biocontrol) agent. An important consideration for any such agent is its target specificity. In this study, the susceptibility to CyHV‐3 of a range of non‐target species (NTS) was tested. The NTS were as follows: 13 native Australian, and one introduced, fish species; a lamprey species; a crustacean; two native amphibian species (tadpole and mature stages); two native reptilian species; chickens; and laboratory mice. Animals were exposed to 100–1000 times the approximate minimum amount of CyHV‐3 required to cause disease in carp by intraperitoneal and/or bath challenge, and then examined clinically each day over the course of 28 days post‐challenge. There were no clinical signs, mortalities or histological evidence consistent with a viral infection in a wide taxonomic range of NTS. Furthermore, there was no molecular evidence of infection with CyHV‐3, and, in particular, all RT‐PCRs for viral mRNA were negative. As a consequence, the results encourage further investigation of CyHV‐3 as a potential biocontrol agent that is specific for carp.     

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp,Cyprinus carpio L. using in situ hybridization

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody‐based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin‐embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post‐infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.     

草鱼出血病基因疫苗的免疫效果

为探讨含有草鱼呼肠孤病毒外衣壳蛋白VP6基因的重组基因疫苗对草鱼出血病的免疫效果,将重组基因疫苗分别配制为10、30和60μg三个梯度,同时设30μg pFastBacTMDual空载体组,均以等体积的氢氧化铝佐剂为免疫增强剂,对草鱼进行注射,以未经处理的草鱼作为对照组。定期采血测定抗体效价,最后对各组草鱼进行攻毒试验并计算死亡率和免疫保护力。结果显示：注射疫苗的三组草鱼均产生抗体,10μg组效价为1：6～1：11;30μg组为1：9-1：13;60μg组为1：11-1：17,且各组效价均在第28天测定最高,免疫保护率依次为100％、100％和95％,而空载体组和对照组为70％和0％。研究表明,疫苗能够诱导草鱼产生免疫反应,为草鱼出血病核酸疫苗的生产应用和产业化提供了重要的理论依据。