猪繁殖与呼吸综合征病毒GP5蛋白分段表达及其生物学功能的鉴定

为进一步研究PRRSV GP5蛋白的生物学功能,本研究将已分离的PRRSV北美洲型野毒株TA-12的GP5基因分为4段(79 bp～207bp、133bp～330bp、229bp～492bp和382bp～603bp)分别克隆于pGEX-6p-1中,并转化E.coli BL21(Rosetta)细胞进行诱导表达.表达蛋白经纯化后,以间接荧光方法(IFA)检测它们与Marc-145细胞的相互作用.IFA检测结果表明GST-GP5-1和GST-GP5-2融合蛋白均能够与Marc-145细胞结合,而且其结合位点在27aa～110aa区域.本实验为进一步研究GP5蛋白生物学功能提供试验依据.     

Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains six structural proteins the roles of which are not completely understood. In a preceding study, immunization with the dutch isolate I10 of PRRSV had led to the development of MAbs against four structural proteins [Wieczorek-Krohmer, M., 1994. Herstellung und Charakterisierung von monoklonalen Antik?rpern gegen das Virus des Porzinen Reproduktiven und Respiratorischen Syndroms (PRRSV). Inaugural-Dissertation, Ludwig-Maximilians-Universit?t, München] here finally identified by reaction with individual plasmid-expressed PRRSV proteins as products of ORFs 3 (GP3), 4 (GP4), 5 (GP5) and 7 (N). Surprisingly, the MAbs against GP5 revealed the presence of two antigenically distinct virus populations in the isolate I10, the population PRRSV-'PPV', isolated from plaques and the PRRSV-'EPV', gained by end point dilution. MAbs against GP3, GP4 and N reacted with both I10 populations as well as with natural PRRSV isolates. However, the anti-GP5 MAbs exclusively recognized PRRSV-'PPV'. In this study immunization of mice with both separated I10 populations confirmed that solely PRRSV-'PPV' possesses the property to induce an immune response ultimately leading to the establishment of MAbs against GP5. Whereas the 15 anti-GP5 MAbs (derived from four independent fusions) reacted exclusively with PRRSV-'PPV' of the isolate I10, anti-GP4 MAbs detected their target antigen on various isolates of European origin and were able to neutralize them. As indicated by competition assays and selection of neutralization-resistant virus mutants, all GP5 MAbs are directed against a single antigenic site on the ORF 5 protein. Both groups of neutralizing antibodies bound to the surface of purified virions demonstrating that the recognized epitopes represent surface structures of the virion envelope. However, anti-GP5 MAbs mediated the binding of more gold granules than anti-GP4 MAbs. Comparison of the neutralizing effect of anti-GP4 and anti-GP5 MAbs revealed the anti-GP5 MAbs as the more efficient antibodies. For the complete neutralization of about 100 ID50 of PRRSV-'PPV' anti-GP5 culture supernatant was effective up to a dilution of 1:1280 whereas the most effective anti-GP4 antibodies exhibited a comparable effect only up to 1:64. These results indicate that PRRSV GP5 in principle is a major target for neutralizing antibodies, as is found for other arteriviruses, but that in nature 'ORF 5 escape mutants' may develop as easily as in vitro.     

PRRSV GP5抗原表位的串联表达及生物学活性鉴定

为研究猪繁殖与呼吸综合征病毒(PRRSV)GP5蛋白抗原表位在诊断和基因工程疫苗中的应用,本研究选取(PRRSV)GP5蛋白已鉴定的抗原表位(aa31³7和aa19237和aa192¹98)进行串联后克隆到原核表达载体pGEX-6p-1上,转化E.coli BL21后进行条件优化诱导表达。SDS-PAGE、Western blotting结果表明,该重组目的蛋白(约31 000)得到分泌表达,且具有良好的免疫学活性。     

Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope GP5 glycoprotein.

The objective of the present study was to evaluate the importance of genomic and antigenic variations which may have affected the major envelope glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) isolates responsible for outbreaks in Quebec and Ontario, in comparison with the modified-live U.S. vaccine strain (MLV) and the European prototype strain from Lelystad (LV). Nucleotide sequence analyses of the open reading frame (ORF)5 genes showed that all of the isolates studied were heterogenous, amino acid (aa) identities varied from 88 to 99% with the MLV strain, and between 51 and 54% with the LV strain. The aa substitutions were randomly scattered across the protein, although one region between residues 26 and 39 was found to correspond to a hypervariable region which involved 0 to 3 potential N-glycosylation sites. The ORF5 encoded products of 5 of these isolates, including the MLV and LV strains, were expressed in E. coli as recombinant proteins fused to the glutathione S-transferase (GST) protein and used to raise hyperimmune anti-ORF5 sera in rabbits. The reactivity patterns of strain-specific hyperimmune anti-ORF5 sera and a panel of 4 monoclonal antibodies directed against the ORF5 gene product of the Quebec IAF-Klop strain of PRRSV, indicated that GP5 of field isolates also underwent antigenic variations. The data suggest that neutralizing epitopes, independent of conformation and glycosylation, are also associated with antigenic variability of the GP5 of PRRSV.     

Differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins

The porcine reproductive and respiratory syndrome virus (PRRSV) GP4 and GP5 proteins are two membrane-associated viral glycoproteins that have been shown to induce neutralizing antibodies. In the present study, the host cell gene expression profiles altered by the GP4 and GP5 proteins were investigated by the use of DNA microarrays. Sublines of Marc-145 and HeLa cells were established by stable transfection with open reading frame (ORF)4 and ORF5 of PRRSV, respectively, and differential gene expressions were studied using microarray chips embedded with 1718 human-expressed sequence tags. The genes for protein degradation, protein synthesis and transport, and various other biochemical pathways were identified. No genes involved in the apoptosis pathway appeared to be regulated in GP5-expressing cells. The microarray data may provide insights into the specific cellular responses to the GP4 and GP5 proteins during PRRSV infection.     

高致病性猪繁殖与呼吸综合征病毒HuN4株GP3单克隆抗体的制备及其抗原表位的鉴定

为制备抗高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV) GP3株的单克隆抗体(MAb),本研究将HP-PRRSV HuN4株免疫BALB/c小鼠,以该病毒感染的细胞及真核表达的GP3蛋白为检测抗原,经间接免疫荧光(IFA)筛选获得了一株稳定分泌MAb的细胞株,命名为4G5.抗体亚类鉴定重链类型为IgG1,轻链类型为K.该MAb细胞培养上清及腹水IFA效价分别为1∶256和1∶1 280.IFA结果显示,MAb 4G5能够识别CH-1R、JXA1-R、HP-PRRSV HuN4株及其疫苗株HuN4-F112,而不识别RespPRRS MLV病毒株.Western blot结果显示,4G5与HP-PRRSV HuN4株及原核表达的GP3蛋白均可以反应,表明其针对的抗原表位为线性表位,中和试验结果显示该MAb无中和活性.通过截短表达GP3蛋白鉴定该MAb抗原表位识别序列为74WCRIGHDRCS83.本研究获得的MAb为进一步研究HP-PRRSV GP3蛋白的结构及功能奠定了基础.     

猪繁殖与呼吸综合征病毒GP5a蛋白在大肠埃希菌中的表达

构建高致病性猪繁殖与呼吸综合征病毒(highly pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)HuN4株GP5a蛋白基因的原核表达载体,并将其在宿主菌BL21(DE3)感受细胞中进行表达。以HP-PRRSV HuN4株GP5a蛋白的基因序列为模板,通过RT-PCR扩增获得GP5a蛋白全长基因片段,将其定向克隆到原核表达载体pGEX-6P-1中,构建含有GP5a蛋白基因的重组表达载体pGEX-GP5a;将构建的重组质粒转化宿主菌,经IPTG诱导,表达出目的蛋白;表达蛋白采用Western blot方法检测其反应原性。结果显示,成功克隆出了HP-PRRSV HuN4株GP5a蛋白基因全长,片段序列为156bp;构建的pGEX-GP5a载体经PCR、双酶切、测序鉴定均正确,转化表达宿主菌后经SDS-PAGE检测目的蛋白以包涵体的形式表达,用包涵体纯化法获得该蛋白;Western blot检测表达蛋白的反应原性,结果显示表达蛋白具有良好的反应原性。本试验成功构建了含有HP-PRRSV HuN4株GP5a蛋白基因的原核表达载体,成功表达、纯化得到了GP5a蛋白,为进一步研究GP5a蛋白的功能提供材料。     

猪繁殖与呼吸综合征病毒GP5蛋白研究进展

猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的猪的传染病,对养猪业危害很大.GP5蛋白是PRRSV ORF5基因编码的一个糖基化的囊膜蛋白,具有较好的免疫原性,能够诱导产生中和抗体.因此,GP5蛋白在PRRSV的致病性、诊断、预防与控制等方面研究中具有重要意义,是研制基因工程疫苗的最佳候选基因.近年来对GP5蛋白的研究取得了重要的进展,现就GP5蛋白的特征、免疫作用及疫苗等方面做一综述.     

猪繁殖与呼吸综合征病毒GP5/M蛋白可溶性表达及免疫原性分析

为表达出高可溶性的GP5/M蛋白并检测其免疫原性,本试验将高致病性猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)HN07-1毒株ORF5和ORF6基因序列上GP5和M蛋白的编码核苷酸序列进行密码子偏爱性优化设计,构建了GP5/M融合Grifin、GST、MBP、NusA、Sumo、Thioredoxin、γ-crystallin、ArsC、PpiB、CeHSP17共10种不同可溶性标签的表达重组载体。分别转化至大肠杆菌BL21(DE3)中,IPTG诱导,SDS-PAGE电泳对融合蛋白的可溶性表达进行检测,筛选出高可溶性表达的GP5/M融合蛋白。重组蛋白通过Ni-NTA Agarose亲和纯化,免疫家兔,对获得的兔抗GP5/M血清进行间接ELISA和病毒中和试验。结果显示,成功构建10个GP5/M表达载体,10个标签中,MBP与GP5/M蛋白相融合的可溶性表达效果最好,并获得了高纯度的MBP-GP5/M重组蛋白质;通过免疫家兔,检测重组蛋白具有免疫原性并能刺激机体产生较高水平的中和抗体。结果表明,试验成功建立了稳定获得GP5/M重组蛋白质的方法,初步鉴定了重组蛋白的免疫效力,为PRRSV亚单位疫苗的后续研究及大量制备奠定基础。     

杆状病毒介导猪繁殖与呼吸综合征病毒GP5蛋白在PK15细胞中的表达与鉴定

以pFastBac I为载体骨架,通过PCR从pcDNA3.3-TOPO载体引入巨细胞病毒(cytomegalovirus,CMV3.3)强启动子及不同功能调控元件,构建成含有双启动子,能在PK15细胞(porcine kidney cell)中表达增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)的昆虫杆状病毒转移(穿梭)载体pFastBac I-CMV3.3-eGFP。收获重组杆状病毒,侵染PK15细胞。倒置荧光显微镜下验证该重组转移载体构建成功,根据荧光水平筛选出重组杆状病毒侵染PK15细胞的最佳MOI(multiplicity of infection)和最佳表达强度时间。另将PRRSV(porcine reproductive and respiratory syndrome virus)GP5蛋白基因克隆到pFastBac I-CMV3.3载体。获得的重组杆状病毒以MOI=100侵染PK15细胞48h后收获细胞产物。Western blot鉴定目的蛋白的表达。结果表明,成功构建了重组杆状病毒介导PRRSV GP5蛋白在PK15细胞表达系统。因此,建立的重组杆状病毒在PK15细胞中高效表达GP5蛋白的方法,将为研究PRRSV吸附机制,GP5蛋白与宿主细胞互作蛋白的筛选和针对PRRSV基因工程疫苗的开发奠定了坚实的基础。     

美洲型猪繁殖与呼吸综合征病毒荧光RT-PCR检测方法的建立

参照美洲型猪繁殖与呼吸综合征病毒（PRRSV）M基因序列，设计引物和探针，并建立其荧光RT-PCR检测方法，以检测严重危害我国养猪业的此型PRRSV。该法能检测到1 TCID50的PRRSV。用此法检测猪瘟病毒、乙脑病毒、伪狂犬病毒、马动脉炎病毒、圆环病毒Ⅱ型和MARC145细胞的RNA，结果均为阴性。对126份临床样品进行检测应用，检测结果（7份阳性，119份阴性）与病毒分离完全符合。此法中的阳性对照为体外转录的dsRNA，具有高度的稳定性，解决了此类检测方法所用的RNA阳性对照所普遍存在的易于降解的难题。     

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003–2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003–2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.     

PRRSV GP5蛋白及猪IL-18基因共表达核酸疫苗免疫原性试验

本试验通过分子克隆技术分别构建了单独表达猪繁殖与呼吸综合征病毒（PRRSV）GP5基因以及PRRSVGP5基因和猪IL-18基因共表达的重组核酸疫苗质粒（pEGFP—GP5和pEGFP—ILl8-GP5）,并进行仔猪免疫原性研究,对构建的PRRSV核酸疫苗所诱导的体液免疫和细胞免疫水平进行检测,进一步研究了PRRSV核酸疫苗免疫效果以及猪IL-18基因对PRRSV核酸疫苗免疫调节作用的影响。同时,调查了商业上应用不同类型的PRRSV疫苗诱导的免疫效果,并与核酸疫苗免疫效果进行比较。结果表明,IL-18作为免疫佐剂在疫苗免疫猪后诱导的病毒特异性细胞免疫反应方面具有很好的调节作用,共表达IL18-GP5蛋白能够明显的改善DNA疫苗的免疫效力,增强抗PRRSV的免疫保护。因此,DNA疫苗做为一种新一代疫苗可用于对抗高致病性PRRSV感染。     

高致病性猪繁殖与呼吸综合征病毒变异株主要囊膜糖蛋白GP5的遗传变异分析

为了探究高致病性猪蓝耳病病毒（HP-PRRSV）的遗传变异特征,本研究对GenBank中235株HP—PRRSV GP5序列的遗传进化、主要氨基酸基序、抗原性以及N-糖基化位点数量和位置的变异进行了分析。结果表明HP-PRRSV之间的同源性较高,与其他参考毒株相比,这些病毒处于一个相对独立的分支中,而且与经典疫苗株的亲缘关系较远。这有助于解释经典疫苗株对于HP—PRRSV为何起不到理想的免疫保护效果。在病毒的中和表位序列中存在着规律的点突变,同时抗原性比较显示HP-PRRSV与经典毒株之间存在着一定的差异。绝大多数的HP-PRRSV的N-糖基化位点在数量上多一个,而且位置要向羧基端平移2个氨基酸,从而使得中和表位两侧直接与糖链相连,可能会造成中和表位被糖侧链所遮掩,本研究由此推测减弱中和抗体对HP-PRRSV的有效识别,促进病毒逃避机体体液免疫。     

猪繁殖与呼吸综合征病毒国内分离毒株GP3、GP5和N蛋白抗原性分析

本研究利用抗美洲型病毒株的GP3、GP5和N蛋白的11株单克隆抗体,对国内分离毒株的GP3、GP5和N蛋白的抗原表位进行鉴别,并同国外参考毒株VR-2332、NVSL(美洲型,B型)、Lelystad(欧洲型,A型)及商品化疫苗毒的抗原多样性进行了比较与分析.通过对免疫过氧化物酶单层试验改进,建立了免疫荧光单层试验(IFMA),并采用该方法对国内分离毒株的GP3、GP5和N蛋白的抗原特性进行了鉴定,研究结果发现国内分离毒株和北美洲型VR-2332株的抗原性比较相近,而与欧洲型LV株的GP3、GP5的抗原性差异较大.根据N蛋白的单克隆抗体(ISU15A-E)与国内分离毒株的反应谱,按照美国L.Yang等[3]分类方法,本试验中的国内分离毒株均属于Ⅰ组.     

Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets

Pseudorabies virus (PRV) has been developed as a vaccine vector for expressing foreign immunogens. Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), continues to be a major problem to the pork industry worldwide. Many vaccine strategies have been developed to control the disease but most of them turn out to be unsuccessful. The objective of this research was to explore the feasibility of PRV-based vector vaccine in protection against PRRSV. A live attenuated vaccine-based PRV recombinant expressing the envelope protein GP5 of PRRSV was generated using recombinant DNA techniques. The Bartha-K61-derived recombinant virus, named rPRV-GP5, was shown to express PRRSV GP5 efficiently. Sixteen healthy piglets were assigned to one of four groups (one to four, four pigs per group). Animals in Groups 1 and 2 were each inoculated intramuscularly and intranasally with 10(7.0) PFU of rPRV-GP5 and its parent Bartha-K61, respectively; Group 3 were vaccinated intramuscularly with one-dose of PRRS inactivated vaccine; Group 4 was served as non-vaccinated control. One month later, all animals were all challenged with 10(6.5) TCID(50) of virulent PRRSV CH-1a. All animals in Groups 1 and 3 remained clinically healthy before and after challenge, with only a short period of fever (no more than 41 degrees C and 3 days), mild and gradually improving lung and kidney lesions, and short-term viremia (2 and 3 week, respectively) in spite of no detectable anti-PRRSV antibody before challenge. On the other hand, all animals in the other two groups showed evident clinical signs with higher temperatures (more than 41 degrees C) after challenge, and severe lung, kidney and spleen lesions and extended viremia (4 weeks). The results indicate that the rPRV-GP5 is safe for vaccinates and able to confer significant protection against clinical disease and reduce pathogenic lesions induced by PRRSV challenge in vaccinated pigs.     

PRRSV JL/07/SW毒株GP5基因克隆与真核表达

根据PRRSV VR-2332毒株核苷酸序列,设计针对GP5基因的特异引物,以PRRSVJL/07/SW毒株细胞培养物为模板,利用RT-PCR方法,成功的扩增出GP5基因,将该基因与真核表达载体pCI-neo连接,转染Marc-145细胞中,用间接免疫荧光试验、SDS-PAGE和western-blot鉴定目的蛋白的表达。结果:RT-PCR分别扩增出GP5基因大小与预期一致,与国际PRRSV美洲型VR-2332毒株的GP5基因序列同源率为89.2%;间接免疫荧光、SDS-PAGE及Western blot试验证明GP5蛋白在Marc-145细胞中成功表达;同时利用pCI-neo真核表达载体在Marc-145细胞中成功表达了PRRSVJL/07/SW毒株GP5基因,为下一步研究蛋白的功能奠定了基础。     

Highly pathogenic porcine reproductive and respiratory syndrome virus GP5 B antigenic region is not a neutralizing antigenic region

In 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) caused great economic losses emerged in China and continues to be a threat for the pig industry. B antigenic region (AR) ((37)SHL/FQLIYNL(45)) of GP5 was considered to be a major linear neutralizing AR in PRRSV classical strains. However, peptide-purified antibodies against this AR did not neutralize PRRSV in a recent report. Compared with classical PRRSV, one amino acid mutation (L/F(39)→I(39)) was found in B AR of HP-PRRSV. To study the ability of B AR of HP-PRRSV to induce neutralizing antibody (NA) in vitro and in vivo, rabbit antisera against B AR with and without the mutation and pig hyperimmune sera with high titer of NAs against HP-PRRSV were prepared. Immunofluorescence assays (IFA) showed that the two rabbit antisera both had reactivity to classical PRRSV CH-1a and HP-PRRSV HuN4 with no observable difference in IFA titer. However, antisera did not have neutralizing activity against classical PRRSV CH-1a and HP-PRRSV HuN4. No correlation was observed between the levels of anti-B AR peptide antibodies and NAs in pig hyperimmune sera that were detected by indirect ELISA and virus neutralization, respectively. B AR peptide-specific serum antibodies had no neutralizing activity and, GST-B fusion protein could not inhibit neutralization of NAs in pig hyperimmune sera. Based on these findings, we conclude that B AR of HP-PRRSV is not a neutralizing AR of HP-PRRSV GP5.     

PRRSV JL／07／SW毒株GP5基因克隆与原核表达

根据GenBank发表的猪繁殖与呼吸综合征病毒(PRRSV)VR-2332毒株核苷酸序列,设计了扩增PRRSV JL/07/SW毒株GP5基因的1对引物,利用RT-PCR方法,从PRRSV JL/07/SW毒株的细胞培养物中成功的扩增出GP5基因,其中GP5基因全长603 bp,与国际PRRSV美洲型VR-2332毒株的GP5基因序列同源率为89.2%;随后,将该基因与原核表达载体pGEX-4T-1连接,转化到宿主菌BL21中进行表达.结果证实PRRSV JL/07/SW毒株GP5基因成功地进行了原核表达,表达的目的蛋白占菌体总蛋白含量的22.8%.