非典型犬瘟热病毒核衣壳蛋白基因序列分析及其在大肠杆菌中的表达

为分析当地非典型犬瘟热病毒(CDV)核衣壳蛋白(N)基因的序列特征及其表达产物的抗原性,根据已发表CDV的N基因序列设计引物,用RT-PCR方法从引起非典型症状的CDV细胞培养物中扩增N基因,进行克隆和序列分析,结果表明:该非典型CDV的N基因与已发表的12个CDV强毒株的核苷酸序列和氨基酸序列同源性分别在96.6%～99.2%和97.9%～99.4%之间,与已发表的4个CDV疫苗弱毒株的同源性分别在93.2%～93.6%和96.4%～97.5%之间;在N基因系统发育进化树上,非典型CDV与12个强毒株处在同一亚群,而且与9个中国分离毒株的亲缘关系近于3个国外毒株。N基因在大肠杆菌中表达的重组N蛋白的分子量为62 ku,主要以包涵体的形式存在;用western blot分析,重组N蛋白可与CDV阳性血清发生特异性反应;以纯化的重组N蛋白为抗原建立的CDV抗体间接ELISA检测方法具有良好的特异性。     

Two Akabane virus glycoprotein Gc domains induce neutralizing antibodies in mice

Akabane virus (AKAV), belonging to the genus Orthobunyavirus and family Peribunyaviridae, causes reproductive and congenital abnormalities in ruminants. Its envelope glycoprotein Gc is a neutralizing antigen, on which at least five distinct antigenic regions have been identified. We attempted to identify the domains using truncated recombinant AKAV Gc proteins expressed in Escherichia coli and monoclonal antibodies (mAbs) with AKAV-neutralizing activity. Dot blot analysis revealed that amino acid positions 1–97 and 189–397 (Gc_1–97 and Gc_189–397) in the truncated recombinant proteins reacted with the mAbs. Additionally, AKAV was neutralized by sera from mice immunized with these recombinant proteins. The results suggested that the two domains contain neutralizing epitopes and could be potential subunit vaccines against AKAV.     

Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus

Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A.     

IBV AH1-99分离株N基因的克隆及序列分析

利用RT-PCR技术成功扩增出IBV AH1-99分离株的N基因全长cNDA,将其克隆到pMD18-T载体上。经酶切分析、PCR鉴定及核苷酸序列测定,成功获得该分离株N基因的重组质粒。序列分析结果表明,分离株N基因全长1 230个核苷酸,编码409个氨基酸。同部分IBV参考毒株相比,核苷酸同源性为85.8%~89.9%,氨基酸同源性为86.6%~91.0%,在进化关系树中,AH1-99与国内分离株X、LX4亲缘关系较近。     

地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

番茄黄化曲叶病毒(TY)侵染性克隆接种鉴定方法研究

采用YEP1和YEP2培养基培养番茄黄化曲叶病毒侵染性克隆pBinPLUS-1.7A＋2β,并用固体菌落、液体菌菌液浓度OD600≈0.6、液体菌菌液浓度OD600≈1.0对感病番茄品种MM、中蔬4号、98-B1在4~6叶苗期进行接种处理。结果显示,2种液体培养基的液体菌菌液浓度为OD600≈0.6时接种处理发病率较高,并初步确立了无TY发病地区侵染性克隆接种鉴定方法。     

两株新分离的APMV-I毒株感染SPF鸡的超微病理变化

将APMV-I鹅源分离株YG97鸡源分离株Y98和NDV强毒株F48E8感染SPF鸡，运用透射电镜观察该2株病毒分离株对机体细胞的影响，结果显示3株禽副黏病毒均引起机体肝、胰、脾、肾、心、胃、十二指肠和直肠等实质器官组织细胞的超微病变，如黏膜上皮细胞多处受损，表面微绒毛脱落；实质细胞核固缩、凝聚、凹陷、核多形性，线粒体嵴断裂、空泡样变、膜溶解，粗面内质网扩张、囊泡变，细胞浆的囊泡内及细胞浆内存在有囊膜的成熟病毒粒子等，但YG97和Y98对不同实质器官的组织细胞超微病变程度不一：胃、肝、胰、脾、肾超微病变比心脏的超微病变严重。电镜观察结果还表明：3株禽副黏病毒引起宿主细胞的超微病变有2种形式：坏死性病变和凋亡性病变。     

新疆地区蚊类携带西尼罗病毒情况调查研究

为查明新疆地区蚊体内携带西尼罗病毒的情况,从2005年-2007年每年的7月~10月采集新疆不同地区的蚊子共计16000余只,并提取总RNA,通过RT-nest PCR的方法检测样品中西尼罗病毒。结果表明,调查样品中没有检测到西尼罗病毒,但不能就此推测新疆是否存在西尼罗病毒,至少对新疆地区和全国制定针对不同动物和人的积极防御政策有重要的公共卫生学意义。     

甘薯褪绿矮化病毒RNase3蛋白的原核表达及抗血清制备

以本实验室保存的重组质粒为模板,利用PCR方法扩增获得了甘薯褪绿矮化病毒SPCSV的RNase3基因,RNase3基因由690个核苷酸组成,编码229个氨基酸。将RNase3基因克隆到原核表达载体pET-28a(+),转化大肠杆菌BL21(DE3),经IPTG诱导,对诱导产物进行SDS-PAGE分析。结果表明,RNase3在大肠杆菌中能高效表达,融合蛋白分子量约为26.5kD。以表达的融合蛋白为抗原,免疫家兔,制备了SPCSV-RNase3的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清对RNase3融合蛋白的效价达10万倍。