蓝舌病病毒基因产物及其生物学功能

蓝舌病病毒(bluetongue disease virus,BTV)是呼肠孤病毒科环状病毒属的双股RNA病毒,其核酸由3个大片段(L1～L3)、3个中片段(M4～M6)和4个小片段(S7～S10)等10个节段组成,分别编码7种结构多肽(VP1～VP7)和4种非结构多肽(NS1、NS2、NS3a、NS3b)。通过dsRNA基因组进行体外翻译,再根据各基因与所编码蛋白的关系,查明了各基因编码的蛋白质及其分子质量和功能。     

蓝舌病病毒分子生物学研究进展

蓝舌病是一种虫媒病毒性传染病,能引起反刍动物的高热,白细胞减少,口。舌、唇和胃肠道粘膜水肿、发组、充血、淤血坏死等炎性反应,孕畜可引起流产。该病最早发现于南非,目前在非洲、美洲、欧洲、亚洲及大洋洲的一些国家均有发生。我国有些省市和地区也有发生,造成严重经济损失蓝舌病病毒是呼肠孤病毒科环状病毒属的典型病毒,为分节段的双链ＲＮＡ病毒,目前至少有２４个血清型。由于基因序列重排和点突变现象的存在,还可能出现新的血清型。病毒颗粒呈圆形,２０面体对称,直径５０ｎｍ—６０ｎｍ,具有双层衣壳,外衣壳由ＶＰ２和Ｖ…     

抗蓝舌病病毒VP7和NS2蛋白单克隆抗体的制备及鉴定

为建立蓝舌病病毒(BTV)的检测方法和研究该病毒蛋白的功能,本研究利用BTV血清8型(BTV8)免疫BALB/c小鼠,取免疫后的小鼠脾淋巴细胞与SP2/0细胞融合,制备单克隆抗体(MAb).并以BTV8作为包被抗原建立间接ELISA方法,经筛选获得了8株稳定分泌抗BTV8 MAb的杂交瘤细胞株(1B2、1F6、2B1、2D10、3B6、3D9、4D4和4D12).Western blot结果显示,MAb 1F6、2B1、2D10、3B6、3D9与BTV8 VP7蛋白反应,MAb B2、4D4、4D12与BTV8 NS2蛋白反应.间接免疫荧光结果显示,该8株MAb与24型BTV血清型呈不同的反应论系.本研究所获得的MAb为建立BTV免疫学检测方法和相关病毒蛋白的功能研究提供了实验依据.     

抗蓝舌病病毒VP6和VP7蛋白单克隆抗体的制备及鉴定

为制备针对蓝舌病病毒(BTV)的单克隆抗体(MAb),本研究利用血清型1型BTV(BTV1)免疫BALB/c鼠,将其脾淋巴细胞与SP2/0进行融合,并用BTV1包被ELISA板,通过间接ELISA方法筛选出3株稳定分泌抗BTV1的MAb的杂交瘤细胞株(2B10、3D4和4H8)。利用表达BTV1主要蛋白的真核表达重组质粒转染BHK-21后,对所制备的杂交瘤细胞株上清进行间接免疫荧光(IFA)以及western blot鉴定,结果显示:2B10和4H8与VP7蛋白反应,而3D4与VP6蛋白反应。同时,IFA鉴定结果进一步表明,3株MAb与24个血清型的BTV均可以发生反应。本研究制备的MAb为建立BTV免疫学检测方法和相关病毒蛋白的功能研究奠定了基础。     

蓝舌病研究综述

蓝舌病是由蓝舌病病毒引起的一种传染病,主要通过库蠓叮咬在反刍动物和骆驼中间传播流行。本文对该病的流行历史,传播,病原学,临床症状,诊断方法以及防控措施进行了综述。     

Epitope Mapping and Immuno-detection of VP7 Protein of Bluetongue Virus Serotype 1

In this study,in order to map the key amino epitope of VP7 protein of blue tongue virus serotype 1 (BTV1),we used monoclonal antibody against VP7 protein of BTV1 to screen 7 mer phage display random peptide library. The selected phages including VP7 protein consensus sequence were amplified and purified,immunoreactivity between epitope of selected phages and monoclonal antibody against VP7 of BTV1 was analyzed by ELISA and competitive ELISA (c-ELISA).The result showed that the epitope of phages including LNWPMVR sequence could specially combine monoclonal antibody against VP7 of BTV1,and the combination could be inhibited or blocked by BTV1,it demonstrated that 7 mer phage simulated the antigenic determinant of BTV protein which could combine monoclonal antibody against VP7 of BTV1.The results suggested that the amino acides 163 to 189(LNAGARGDVQQIFQGRNDPMMLYLVWR) were the specific epitope of VP7 protein of BTV.     

蓝舌病1型病毒VP7蛋白表(拟)位定位及免疫检测分析

为研究蓝舌病1型病毒VP7蛋白关键性氨基酸表位定位,本试验应用抗蓝舌病1型病毒VP7蛋白单克隆抗体淘选噬菌体展示的7肽随机肽库,对筛选的共有序列噬菌体扩增纯化,通过ELISA、竞争性ELISA分析共有噬菌体拟位与蓝舌1型病毒VP7蛋白单克隆抗体的免疫反应性。结果表明,含有LNWPMVR基序的噬菌体拟位能够与蓝舌病1型病毒VP7蛋白单克隆抗体发生特异性结合,并且此结合能被蓝舌病1型病毒抑制或阻断,表明噬菌体7肽模拟了BTV蛋白上与蓝舌病1型病毒VP7蛋白单克隆抗体结合的抗原决定簇,提示蓝舌病病毒VP7蛋白的第163-189位氨基酸（LNAGARGDVQQIFQGRNDPMMIYLVWR）构成蓝舌病病毒VP7蛋白特异性表位。     

蓝舌病病毒VP7蛋白的原核表达及免疫原性鉴定

为获得蓝舌病病毒(bluetongue virus,BTV)25型的VP7原核表达蛋白,本试验以蓝舌病病毒重组质粒为模板,PCR扩增VP7基因,将其克隆于pET-24b(+)表达载体中,获得pET-24b-BTV-VP7重组质粒。经酶切和测序鉴定后,转化大肠杆菌BL21(DE3)受体菌,IPTG诱导表达His-BTV-VP7蛋白。在变性条件下用镍亲和层析柱纯化His-BTV-VP7蛋白,经Western blotting及ELISA鉴定其免疫原性。结果显示,His-BTV-VP7蛋白以包涵体形式表达,大小约为40 ku;Western blotting和ELISA检测此原核表达蛋白能与山羊阳性血清发生特异性反应,具有良好的免疫原性。本研究为后续建立蛋白芯片检测方法奠定了基础。     

Prokaryotic Expression and Immunogenicity Identification of Bluetongue Virus VP7 Protein

To obtain VP7 protein of bluetongue virus (25 type), VP7 gene was amplified and cloned in pET-24b(+) expression vector.The pET-24b-BTV-VP7 recombinant plasmid was transformed into BL21 (DE3), then the VP7 protein of bluetongue virus was expressed using IPTG and purified by nickel affinity chromatography in vitro.Immunogenicity of VP7 protein was determined by Western blotting and ELISA.The results showed that the molecular weight of VP7 protein was about 40 ku and it could react with goat positive serum specifically.This study laid the foundation for establishing protein chip detection methods in the future.     

动物蓝舌病毒L2基因克隆及序列分析比较

对 15株中国动物蓝舌病毒血清 1型 (BTV- 1)野毒株及 1株弱毒疫苗株 L 2基因进行了克隆和测序 ,并进行了系统进化分析。中国 BTV- 1型毒株分为 2个组 :分离年代最早的 Y86 3毒株 (1979年 )为 组 ;1996年以后分离的 14株毒株为 组。 2组之间的同源性是 91.6 %。 组又分为 4个谱系 ,疫苗株 Vac F45与原始株 V6 5 8分别属于 组的第 1和第 3谱系 ,核苷酸同源性 98.6 %。由此表明 ,中国 BTV- 1型毒株在自然进化及鸡胚传代驯化过程中 ,L2基因发生了遗传变异。     

蓝舌病病毒血清1型野毒株及疫苗株S10基因差异

蓝舌病病毒血清型 1型是主要致病血清型之一。为明确其流行规律的分子生物学基础 ,采用 RT- PCR扩增和序列测定技术分析了 15株野毒株及 1株弱毒疫苗株的 S10全基因片段 ,并对其核苷酸和氨基酸差异进行了比较。所有毒株 S10基因核苷酸长度均为 82 2 bp,含有 2个起始密码子 (核苷酸 2 0～ 2 2和 5 9～ 6 1)和 1个终止子 (核苷酸 70 7～70 9) ,预测编码 2种蛋白 (NS3和 NS3A)。不同毒株间 S10基因核苷酸差异为 0～ 138个 (同源性 10 0 %～ 82 %) ,NS3/NS3A蛋白氨基酸差异为 0～ 15个 (同源性 10 0 %～ 93%)。基于 S10基因序列分析 ,可将蓝舌病病毒野毒株及疫苗株分为 2个基因群 :12株野毒株及 1株疫苗株为基因 群 ,3株野毒株与澳大利亚 型毒株属于基因 群 ,两群间的核苷酸同源性为 79%。各基因群在地域分布及宿主来源上未发现有明显的特征性。基因群与毒株分离年代、对 BHK- 2 1细胞毒力等特征关系亦不明显。     

Establishment and Preliminary Application of Indirect ELISA Based on Recombinant NS4 Protein for Detection of Bluetongue Virus Antibodies

This study was conducted to establish an indirect ELISA method for the detection serological antibody of bluetongue virus (BTV). The purified BTV recombinant NS4 protein obtained from the prokaryotic express system was used as the coated antigen, and then an indirect ELISA antibody detection method of BTV was developed by optimizing the reaction conditions. SDS-PAGE results showed that the recombinant NS4 protein with a size of about 52 kDa was obtained, which mainly existed in the supernatant. Western blot results showed that the purified recombinant NS4 protein had good antigenicity. The ELISA reaction conditions were optimized by the square matrix test. The optimal coating amount of recombinant NS4 protein antigen was determined to be 3.0 μg per well, and the optimal dilution ratio of serum to be tested was 1:200, and the optimal dilution concentration of HRP-labeled rabbit anti-cow IgG secondary antibody was 1:4 000, and the critical values were 0.29 and 0.35, respectively. The detection sensitivity of the BTV antibody was up to 1:1 600. The intra-assay repeatability and the inter-assay repeatability coefficient of variation were less than 10%. The positive coincidence ratio and negative coincidence ratio were 98% and 100% respectively. The indirect ELISA method established in this study laid a foundation for clinical serum antibody detection and serum epidemiological investigation of BTV.     

基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用

旨在建立蓝舌病病毒（BTV）血清学ELISA抗体检测方法,本研究以原核表达并纯化的BTV NS4重组蛋白为包被抗原,通过反应条件优化,建立了一种BTV重组NS4蛋白的间接ELISA抗体检测方法。SDS-PAGE结果显示,获得大小约52 ku的NS4重组融合蛋白,主要在上清中存在,Western blot显示,纯化后的重组蛋白具有良好的抗原性。通过方阵试验进行了ELISA反应条件优化,确定了重组蛋白抗原最佳包被量为3.0 μg·孔^-1;血清最佳稀释倍数为1：200,酶标二抗最佳工作浓度为1：4 000,临界值分别为0.29和0.35。上述以NS4蛋白作为包被抗原建立的BTV抗体间接ELISA方法检测敏感性可达1：1 600;批内和批间重复性变异系数均小于10%;检测76份重庆地区牛群血清样品,阳性符合率为98%,阴性符合率为100%。本研究建立的间接ELISA方法为临床BTV血清抗体检测及BTV血清流行病学调查奠定了基础。     

Prokaryotic Expression and Immunoreactivity Analysis of VP2 Protein of Bluetongue Virus Serotype 1

The study was aimed to test the immunoreactivity of the VP2 protein of bluetongue virus serotype 1 (BTV-1) in vitro. Based on the published BTV-1 L2 gene of Y863 strain, specific cloning PCR primers were designed and synthesized. The L2 gene was amplified through RT-PCR method and then was purified and cloned into the expressing vector pEASY-Blunt E1. The cloned recombinant plasmids were identified. The positive recombinant L2 plasmid was cloned into BL21(DE3) competent cells to express VP2 protein. The acquired purified recombinant BTV-1 VP2 protein was analyzed through the methods of Western blotting, ELISA and blocking ELISA. The results showed that:BTV-1 VP2 protein was expressed as the inclusion bodies in the pEASY-Blunt E1 vector; 160 and 200 mmol/L glyoxaline were the best condition to wash down the expressed protein. The molecular weight of this purified recombinant protein with N-terminal His-tag was about 105 ku. Through the results of Western blotting, ELISA and blocking ELISA, it had been proved that this recombinant protein could combine with BTV-1 specific antibody and this combination could be blocked by BTV-1 virus. The study showed that the recombinant BTV-1 VP2 protein, expressed through the prokaryotic expression vector pEASY-Blunt E1, possessed good immunoreactivity and this study had established foundation for locating the serotypic epitopes of the BTV-1 VP2 protein.