摘要
蓝舌病病毒(Bluetongue virus,BTV),属呼肠孤病毒科、环状病毒属、蓝舌病病毒亚群(Bluetongue virus subgroup),该病是由库蠓传播的非接触性反刍动物病毒性传染病,纯种细毛羊对该病最为敏感,死亡率高达35%,世界动物卫生组织(OIE)将其列为A类疫病,并且要求实时通报疫病,我国已将其规定为一类动物传染病。迄今为止尚未发现BTV对人类有传染性。
BTV最早发现于1876年,呈世界性分布。目前已从非洲、欧洲、亚洲、北美、南美和大洋洲的多个热带、亚热带和温带地区国家分离到BTV,并且其分布范围不断扩大,危害也日趋严重。我国于1979年发现本病存在,现已从全国29个省检出BTV血清阳性家畜。到目前为止,全世界共分离到26个血清型BTV,我国已鉴定出BTV-1、2、4、9、15、16、23等7个血清型,其中BTV-1和BTV-16是主要的致病血清型。
BTV基因组大小约19Kb,由10个分节段的双股RNA组成,编码7种结构蛋白(VP1～VP7)和5种非结构蛋白(NS1,NS2,NS3,NS3a,NS4)。VP7蛋白由S7基因编码,大小为349个氨基酸,位于病毒核衣壳的表面,约占病毒核心蛋白总量的36%。研究表明,各血清型BTVVP7蛋白94%以上的氨基酸保守,是BTV群特异性抗原蛋白。目前,国际上已经开展了一些针对于蓝舌病病毒VP7蛋白抗原表位鉴定的研究工作,但主要还是针对抗原线性表位的鉴定,对于VP7蛋白构象表位的鉴定工作还没有报道。Grimes等人构建的晶体结构表明,VP7蛋白呈三聚体结构,其中第134～253位氨基酸所构成的晶体结构位于VP7蛋白三聚体结构的外表,是决定VP7蛋白抗原性的主要部位,也是与宿主细胞识别并吸附的主要部位。
由于BTV血清型众多,各血清型之间无交叉保护作用。在这种情况下,加强蓝舌病病原学监测工作,使用准确的检测手段对病原进行检测,及时隔离和处理已感动物,是减少经济损失的关键。以具有群特异性阻断效果的单克隆抗体(McAb)为基础的竞争ELISA(C-ELISA)检测待检样品中的BTV抗体,具有特异、敏感、稳定和安全的特点,是OIE指定的血清学检测方法之一,但由于进口免疫学检测试剂盒价格昂贵,无法在畜牧业养殖业中推广应用,所以筛选制备具有BTV群特异性阻断效果的McAb,自主研发C-ELISA检测试剂盒,为我国BT的流行病学调查和监测提供快速、安全及低成本的技术手段工作势在必行。
本研究利用BHK-21细胞对血清型12型BTV病毒进行扩繁,提取病毒RNA,通过RT-PCR方法扩增VP7基因,将扩增后的VP7基因经双酶切后连接到原核表达载体pMAL-c4X上,构建出重组质粒pMAL-c4X-VP7。将重组质粒转化到大肠杆菌TB1感受态细胞中,经IPTG诱导表达MBP-VP7融合蛋白后,利用树脂对该蛋白进行纯化,Western blot和间接ELISA结果表明MBP-VP7蛋白具有良好的BTV群特异性免疫原性,并可诱发机体产生针对于蛋白构象表位的多克隆抗体。
利用纯化后的MBP-VP7蛋白为免疫抗原,免疫BALB/c小鼠,通过杂交瘤技术将免疫后的小鼠脾细胞与骨髓瘤SP2/0细胞进行融合,以血清型12型BTV粒子作为筛选抗原,建立间接ELISA检测方法,筛选分泌VP7蛋白单克隆抗体的杂交瘤细胞,本研究最终获得了5株能稳定分泌针对VP7蛋白的McAb的杂交瘤细胞株,并将其分别命名为：BTV-1F3、 BTV-2D10、BTV-2H10、BTV-4F11和BTV-4H7,这些抗体的获得为BTV检测方法的建立及抗原表位鉴定提供了物质基础。
利用抗体亚类鉴定试剂盒对本研究筛选到的McAb进行鉴定,结果表明,5株McAbs重链均为IgG1,轻链均为K链。间接免疫荧光(IFA)试验表明,单克隆抗体BTV-2D10和BTV-4H7可以与BTV24个血清型发生特异性反应,而与茨城病病毒(IBAV)、中山病病毒(CV)、赤羽病病毒(AKAV)、牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛肠道病毒(BEV)、牛呼肠孤病毒(RV)及口蹄疫病毒(FMDV)不反应,说明这两株McAbs均为BTV群特异性单克隆抗体。竞争ELISA试验证明,BTV-4H7具有良好的BTV群特异性阻断效果,所以本研究以MBP-VP7蛋白作为包被抗原、BTV-4H7作为竞争抗体,建立了C-ELISA方法。利用该方法与IDEXX公司生产的BTV诊断试剂盒进行比较,同时检测15种不同血清型的BTV阳性血清及322份采自广西省不同地区的山羊血清样品,两者的符合率分别为100%和98%,说明该方法可以用于BT的流行病学调查和监测工作,该方法的建立为我国BTV抗体的监测提供了安全、廉价、快速、准确的技术手段。
M13噬菌体展示结果表明,BTV-2D10及BTV-4H7所识别的B细胞表位均为构象表位,其中BTV-2D10所识别的抗原表位至少由四个区域组成,它们分别是VP7蛋白的第185～186位、第205～207位、第236～240位和第278～279位氨基酸所在区域。具有BTV群特异性阻断效果的BTV-4H7所识别的抗原表位同样至少由四个区域组成,它们分别是VP7蛋白的第34～35位、第175-177位、第185～186位和第278～279位氨基酸所在区域;利用生物信息学软件Discovery Studio将M13噬菌体展示的这些氨基酸区域在Grimes等构建的晶体结构模型上进行展示,同时利用DNAStar软件对编码不同血清型BTV VP7蛋白的氨基酸序列进行比对分析,结果表明,利用M13噬菌体展示的这些氨基酸区域在不同血清型BTV VP7蛋白的氨基酸序列上是非常保守的,结果同时表明BTV-2D10有两个识别区域、BTV-4H7有三个识别区域位于VP7蛋白三聚体的LOOP结构上,有研究表明LOOP结构有较强的抗原性,易于刺激机体产生抗体,本研究所得到的结果也证明了这一点。通过软件分析还发现BTV-2D10与BTV-4H7有两个相同的氨基酸识别区域,证明不同的BTV群特异性McAb所识别的抗原表位在结构上有差异,但在氨基酸序列上存在重叠的情况。依据M13噬菌体展示出的构象表位序列,本研究对具有蓝舌病病毒群特异性阻断效果的单克隆抗体BTV-4H7所识别的抗原表位所对应的4个区域的16个氨基酸进行了突变,突变时按照疏水性氨基酸向亲水性氨基酸突变、亲水型不带电荷氨基酸与亲水型带正负电荷的氨基酸相互突变的原则,分别将第33～36位的LGIA突变成NSKT、第174～177位的IFQG突变成KKDS.第183～186位的MIYL突变成QKKN和第278-281位的WHGL突变成KRSN,并将突变后的VP7蛋白进行原核表达,以表达后的蛋白为抗原、以本研究筛选到的McAb为抗体进行间接ELISA试验,鉴定突变后表达的VP7蛋白的抗原性及与BTV-4H7的反应活性。SDS-PAGE结果表明,经过氨基酸突变的VP7基因可以在原核表达系统中进行表达,表达大小与未突变的MBP-VP7蛋白大小相同；利用本研究筛选到的5株McAbs及IDEXX检测试剂盒中的McAb对突变后的VP7蛋白进行间接ELISA验证,结果表明,突变后表达的VP7蛋白可以与本研究筛选到的4株McAbs发生特异性反应,具有较好的抗原性,但却不能与BTV-4H7及IDEXX检测试剂盒中的McAb发生特异性反应,证明M13噬菌体展示的单克隆抗体BTV-4H7所识别的4个区域的9个氨基酸序列是其所识别构象抗原表位的关键氨基酸序列,同时突变后表达的VP7蛋白不能与IDEXX检测试剂盒中的McAb发生特异性反应,这证明在VP7蛋白的氨基酸序列上,具有蓝舌病病毒群特异性阻断功能的核心氨基酸残基有可能是唯一的,这一结果为研究编码VP7蛋白氨基酸的特异性阻断机制奠定了理论基础。
BTV belongs to Reoviridae, Orbivirus, Bluetongue virus subgroup, it was spread by Culicoides, purebred landmark alcala gate was the most sensitive animal for this disease, the mortality was as high as35%. The OIE has listed it as A-Class disease and required report it at anytime, Our country provided it as the first class of animal infectious disease, until now, it has no infectivity to human report.
BTV was firstly discovered in1876. The outbreak of BTV concentrated in tropical, subtropical and temperate countries; it has brought great losses to the Animal husbandry. In China, it was firstly discovered in1979, until now, it has been separated in29provinces. Until now, the whole world has separated26serotypes of BTV. BTV-1,2,4,9,15,16,23were the mostly serotypes in China, among them BTV-1,and16were the mostly pathogenic serotypes.
The genome size of BTV was about19Kb, composed by10parts dsRNA.The genome encoded seven stuctural proteins (VP1to VP7) and four nonstructural proteins (NS1. NS2, NS3, NS3a). VP7protein was encoded by S7gene, composed by349amino acids, it was located on the surface of the virus, VP7protein accounted for36%of the total virus core protein. Relevant research indicated that about94%amino acids of each serotypes BTV VP7protein were highly conserved. The VP7protein was the group-specific antigen of BTV. Grimes etc crystal structure showed that the structure of VP7protein was tripolymer and the function of the top domain (134to253) was antigenic index and combined with the host cell.
Since BTV contains so many serotypes and each country has different serotypes, until now there were no effective vaccines to prevent BTV. So, early diagnosis and prevention were the most effective methods to avoid the disease outbreak. The competitive ELISA was the mostly used method in recent years, it is sensitive, specific, stable and safe features, but the imported kit was so expensive, it cannot be applied in animal husbandry. So it seemed imperative that prepared monoclonal antibodies and established Competitive ELISA method by ourselves.
In this study, the serotype of BTV-12virus was cultivated by BHK-21cell, and the total RNA were extracted, the VP7gene was amplified by RT-PCR method and cloned into prokaryotic expression vector pMAL-c4X. The recombinant plasmid was transformed into E.coli TB1cells and fusion protein (MBP-VP7) produced after induced with IPTG and purified by resin. Western blot and indirect analysis demonstrated that the fusion protein (MBP-VP7) has favorable antigenicity.
In this study, BALB/c mice were immunized by purified MBP-VP7protein, the spleen cells were fused with the SP2/0cells by hybridoma technique, and screened by indirect ELISA coated with purified serotype of BTV-12virus, after3times of sub-clone prepared five strains monoclonal antibody:BTV-1F3,2D10,2H10,4F11and4H7. Subgroup identification results showed that all of the monoclonal antibodies of the heavy chain were IgG1and the light chain were κ. Indirect immunofluorescence test showed that the monoclonal antibodies BTV-2D10and BTV-4H7could occurred specific reaction with24serotypes of BTV but have no reaction with IBAV,CV,AKAV, BVDV, IBRV, BRV, BEV and FMDV. The results indicated that the two monoclonal antibodies were group specific antibodies of BTV.
The competitive ELISA test certificated that the BTV-4H7monoclonal antibody has the BTV group specific blocking effect; therefore, the competitive ELISA method was established by MBP-VP7as the coated antigen and BTV-4H7as the competitive antibody. Clinical tests showed the method has100%agreement with IDEXX kit to detect of15positive serum samples and98%agreement with IDEXX kit to detect of322serum samples from Guangxi. Therefore, the competitive ELISA assay established in this study provided a low-cost, effective, quick and accurate method for the detection of antibodies against BTV.
The results of M13phage display indicated that the epitopes of BTV-2D10and BTV-4H7were conformational,the epitope of BTV-2D10was decided by four regions at least (185to186,205to207,236to240and278to279), the same that the epitope of BTV-4H7was also decided by four regions at least(34to35,175to177,185to186and278to279). The software discovery Studio combined with the crystal structure which constructed by Grimes etc indicated that the epitopes of BTV-2D10has two resions and BTV-4H7has three resions located on the loop structure of the tripolymer,so they were easily produced antibodies. Compared with all serotypes of BTV VP7genes by the software of DNAStar indicated that the epitopes regions were very conservative in different serotypes of BTV VP7genes and the epitopes of two monoclonal antibodies have two same positions indicated that different BTV Group-Specific monoclonal antibodies have overlapped epitope regions.
In order to prove the anthenticity of the M13results, the amino acids of the BTV-4H7epitope were mutanted. In line with nonpolar amino acids to mutate into polar amino acids and exchange of close water type without charge amino acids and close water type with positive and negative charges principles, the amino acids LGIA which were located in the position of33to36were changed to NSKT, the amino acids IFQG which were located in the position of174to177were changed to KKDS, the amino acids MIYL which were located in the position of183to186 were changed to QK.KN, the amino acids WHGL which were located in the position of278to281were changed to KRSN, after that the mutant VP7gene was cloned into prokaryotic expression vector pMAL-c4X, the SDS-PAGE analysis demonstrated that the mutant VP7gene was expressed as same as the natural VP7gene. Indirect ELISA analysis demonstrated that the mutant fusion protein could react with four monoclonal antibodies which producted in this research but could not react with BTV-4H7and the IDEXX's monoclonal antibody, the results indicated that the epitope of BTV-4H7which displayed by M13was believable and the amino acids which have the effect of block were relative only. These results established the theory basis for the specific blocking mechanism of amino acids which coded the VP7protein.
引文
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