猪繁殖与呼吸综合征病毒分子生物学研究进展

猪繁殖与呼吸综合征病毒(PRRSV)是严重危害养猪业的病原,对PRRSV的分子生物学研究进展进行了综述,主要包括PRRSV的基因组结构、病毒的非结构蛋白和结构蛋白及其功能、致病机理及复制与转录等.     

猪繁殖与呼吸综合征病毒分子生物学研究进展

猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(PRRSV)诱发的一种接触性传染病,其症状主要表现为怀孕母猪流产、早产、产死胎、木乃伊胎及成年猪的呼吸道症状。本病自1987年在美国爆发后,给世界养猪业造成巨大损失。近年来,由于其危害性大而引起专家学者的关注,PRRS在分子生物学方面研究者较多。就其病原特性,基因结构,病毒蛋白,分子生物学诊断以及PRRS基因工程疫苗的研究等方面进行论述。     

检测猪繁殖与呼吸综合征抗体的重组N蛋白-乳胶凝集试验的建立

The purified recombinant nuclocapsid protein of PRRSV was used to conjoined with latex,and the conjoined concentration, temperature and time were optimized, a detection method for serum antibodies against Porcine reproductive and respiratory syndrome virus was established. Some tests for the method's sensitivity, specificity and stability were confirmed. About 200 serum samples were detected by using the method, and these samples were also detected by recombinant N protein based ELISA and IDEXX ELISA kit. The result showed that the agreement ratio of the recombinant N protein based LAT with other two methods arrived at 93%. 78%. respectively.     

猪繁殖与呼吸综合征病毒JL株Nsp10的表达及鉴定

为获得猪繁殖与呼吸综合征病毒JL株非结构蛋白Nsp10,以克隆质粒pMD18-T-Nsp10/JL为模板,将Nsp10基因克隆至原核表达载体pET-32a,转化至大肠杆菌BL21(DE3)中诱导表达,对不同温度和IPTG浓度进行优化,以获得Nsp10蛋白表达的最佳条件,采用SDS-PAGE检测目的蛋白的表达情况,并通过Western blotting检测其免疫活性。结果表明,Nsp10基因成功克隆至pET-32a,有分子量约为43.4 kD的融合蛋白获得表达,重组蛋白于诱导4 h后达到高峰,IPTG浓度为0.2-1.2 mmol/L时,重组蛋白表达量无明显差异,Western blotting证实该表达蛋白具有反应原性。     

三带喙库蚊体内猪繁殖与呼吸综合征病毒的分离与鉴定

【目的】调查猪场蚊虫是否能携带猪繁殖与呼吸综合征(PRRS)病毒。【方法】采集发生PRRS疫情的3个养猪场蚊虫样本,采用RT-PCR方法检测PRRS病毒核酸,取阳性蚊虫样本接种Marc-145细胞进行病毒的分离培养,以间接免疫荧光抗体技术和分子克隆技术进行病毒的鉴定。【结果】 养猪场内的蚊虫主要有三带喙库蚊Culex tritaeniorhychus、凶小库蚊Culex modestus、中华按蚊Anopheles sinensis和骚扰阿蚊Armigeres obturbans,其中三带喙库蚊占86.76%;以PRRS病毒N基因引物进行扩增,三带喙库蚊样本呈现阳性反应,而其他蚊种均为阴性。在蚊虫接种的Marc-145细胞中可见细胞融合和空泡形成等细胞病变效应;用抗PRRS病毒N蛋白抗体和羊抗猪IgG(H+L)-FITC进行间接免疫荧光染色,感染细胞呈现黄绿色荧光;以NSP2基因引物进行RT-PCR扩增、克隆与测序,发现库蚊源病毒与相应猪场猪源病毒中相应基因的序列具有较高同源性。【结论】 三带喙库蚊为猪舍优势蚊种,并能携带猪繁殖与呼吸综合征病毒。     

我国猪繁殖与呼吸综合征病毒NT0901分离株分子特征

猪繁殖与呼吸综合征病毒(PRRSV)是目前引起国内外养猪业严重经济损失的重要病原之一,病毒基因和毒力变异较大。PRRSV NT0801株分离自我国发病猪群,毒力较强,但NSP2基因不存在高致病性PRRSV 30个氨基酸的缺失。为了进一步阐明该分离株的分子特征,本研究对该毒株全基因序列进行了测定和分析,结果该毒株基因组全长15 439 bp,其中包含29 nt Poly(A)。与高致病性PRRSV毒株JXA1比较,核酸序列同源性为96.7%,推导的GP3和GP5氨基酸序列同源性分别为97.2%和98.5%,但NSP2基因无30个氨基酸的缺失;与传统型毒株ch-1a比较,推导的GP3和GP5氨基酸序列同源性分别为92.9%和91.5%;基因进化树分析结果显示其介于高致病性和传统PRRSV毒株之间。与其它不同毒力PRRSV分离株基因序列比较,未发现明显重组信号。不同毒力毒株氨基酸残基比对分析结果显示,15个位点潜在毒力相关氨基酸残基中,该毒株有9个与高致病性PRRSV毒株一致,3个与高致病性PRRSV毒株不同,但与传统型和JXA1疫苗株相同,1个位点只与JXA1疫苗株相同,2个与其它毒株都不相同。表明该分离株与高致病性PRRSV密切相关,PRRSV流行毒株变异与基因突变有关,从而为该病毒毒力基因定位研究奠定了基础。     

猪繁殖与呼吸综合征病毒和盖他病毒双重RT-PCR检测方法的建立与应用

盖他病毒(Getah virus,GETV)可感染猪并引起和猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)相类似的临床表现,且两者可混合感染而难以区分。我国检测PRRSV和其他诸多病毒的多重PCR文献已比比皆是,但尚无同时检测PRRSV和GET V方法的报道。为此,作者根据GenBank中公布的PRRSV和GETV全基因序列,设计了两对分别扩增PRRSV ORF7基因和GETV Cap基因片段的引物,通过反应条件的优化及特异性、灵敏性和重复性试验,首次建立了可同时检测PRRSV和GETV的双重RT-PCR方法。结果显示,该方法可以同时扩增出PRRSV 633 bp和GETV 316 bp的特异性片段,而对猪瘟病毒等其他相关病毒扩增结果均为阴性。对GETV和PRRSV的cDNA最低检测量分别为2.48×10~(-4)ng和2.50×10~(-5)ng。用建立的双重RTPCR方法对河南15个地市92个猪场的136份临床疑似PRRS发病猪样品进行检测,共从58个猪场检出PRRSV单一阳性样品44份,阳性率为32.4%;GETV单一阳性样品7份,阳性率为5.15%;GETV和PRRSV混合感染样品8份,阳性率为5.88%。检测结果与单一RT-PCR结果吻合率达100%。该方法为GETV和PRRSV的快速检测、鉴别诊断和分子流行病学研究等提供了新的技术手段。     

猪繁殖与呼吸综合征病毒实时PCR检测方法的建立

设计合成了一套引物和TaqMan探针,以扩增猪繁殖与呼吸综合征病毒的核衣壳蛋白基因,通过反应条件的优化,在国内首次建立了快速定量检测猪繁殖与呼吸综合征病毒的实时PCR方法,并用该法检测患病猪的肺脏等样品.结果表明该方法具有较好的特异性和重复性,对PRRSV细胞培养物的检测下限为0.01TCID50,敏感性比常规RT-PCR高100倍;对10份PRRS疑似猪肺脏样品检测5份为阳性,与病毒分离的阳性符合率为100%.该方法具有快速、灵敏、准确、低污染等优点,在PRRSV的早期检测、预防控制、进出口检疫及基础研究中会起到重要作用.     

猪繁殖与呼吸综合征病毒实时PCR检测方法的建立

设计合成了一套引物和TaqMan探针,以扩增猪繁殖与呼吸综合征病毒的核衣壳蛋白基因,通过反应条件的优化,在国内首次建立了快速定量检测猪繁殖与呼吸综合征病毒的实时PCR方法,并用该法检测患病猪的肺脏等样品。结果表明该方法具有较好的特异性和重复性,对PRRSV细胞培养物的检测下限为0.01TCID50,敏感性比常规RT-PCR高100倍;对10份PRRS疑似猪肺脏样品检测5份为阳性,与病毒分离的阳性符合率为100%。该方法具有快速、灵敏、准确、低污染等优点,在PRRSV的早期检测、预防控制、进出口检疫及基础研究中会起到重要作用。     

美洲型与欧洲型猪繁殖与呼吸综合征病毒Nsp7蛋白的截短表达和鉴定

猪繁殖与呼吸综合征病毒(PRRSV) Nsp7蛋白具有较强的免疫原性,且在美洲型(NA)和欧洲型(EU) PRRSV之间免疫原性差异显著,是抗体分型检测的理想靶抗原.本研究采用原核表达系统分别表达和纯化了NA和EU PRRSV Nsp7蛋白,Western blotting分析表明重组蛋白与相应血清型抗体有较强的免疫反应,但特异性较差,与另一血清型抗体仍存在一定的免疫反应,预示两种血清型PRRSV Nsp7蛋白存在免疫交叉反应抗原表位,全长表达不能用于分型诊断.利用生物学软件分析NA和EU PRRSV Nsp7的相似抗原表位,采用融合PCR缺失其编码序列后,经表达纯化获得NA-△Nsp7和EU-△Nsp7两种截短蛋白,蛋白大小约为43 kDa,Western blotting分析表明NA-△Nsp7和EU-△Nsp7可分别与相应血清型抗体发生特异性反应,且无免疫交叉反应,为NA和EU PRRSV分型抗体检测试剂的研制奠定了基础.     

重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞

【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。     

Application of GP5 protein to develop monoclonal antibody against porcine reproductive and respiratory syndrome virus

In this study,a panel of monoclonal antibodies (mAbs) against Porcine reproductive and respiratory syndrome virus(PRRSV),named as 8C9 and4B4,were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV (TCID50=5.5),screened by the indirect ELISA and subjected to several limiting dilutions.mAbs were then identified by biological characterization.Among the two fusion cell strains,8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass.The titers in cell culture supernatant and abdomen liquor reached to 1:104and 1:105,respectively.The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively,and no reaction with Classical swine fever virus (CSFV) or Swine vesicular disease virus (SVDV).The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa,respectively.In neutralization activity tests,the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1:512,but mAB 8C9 has no neutralization activities to PRRSV.     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK-/gE-/GP5+expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV),based on the PRV genetically depleted vaccine strain TK-/gE-/LacZ+,scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene (the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK-/gE-/LacZ+,resulting in a PRV recombinant named TK-/gE-/GPSm+,which expressed the modified GPSm protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK-/gE-/GPSm+and TK-/gE-/GP5+expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK-/gE-/GPSm+against PRRSV were higher than that induced by TK-/gE-/GP5+.Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.     

NADC30-like猪繁殖与呼吸综合征病毒样颗粒的制备与鉴定

【背景】猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)可以造成怀孕母猪的繁殖障碍及仔猪的呼吸系统疾病,近年来,NADC30-like谱系PRRSV已成为国内的优势流行毒株。【目的】研制针对NADC30-like谱系PRRSV的病毒样颗粒(virus-like particle,VLP)疫苗。【方法】将PRRSV NADC30-like毒株编码GP5蛋白开放阅读框5(open reading frame 5,ORF5)、ORF6(编码M蛋白)分别连接至pFastBac^TMDual载体P₁₀和P_H启动子下游多克隆位点,获得穿梭质粒pFB-30-ORF5及pFB-30-ORF6,酶切鉴定后,将ORF6基因插入到穿梭质粒pFB-30-ORF5 P_H启动子下游,构建穿梭质粒pFB-30-ORF5-OPF6。将上述3种穿梭质粒分别转化大肠杆菌DH10Bac感受态细胞,通过蓝白斑筛选及PCR鉴定重组杆粒。再将获得的重组杆粒转染至SF9昆虫细胞,发现细胞病变后收获病毒液,继续盲传3代,在透射电镜下观察是否有病毒样颗粒。用第3代病毒液感染SF9细胞后,分别用GP5蛋白、His-tag、Flag-tag单克隆抗体作为一抗,通过免疫电镜、间接免疫荧光(indirect immunofluorescence assay,IFA)、Western blotting鉴定重组蛋白。【结果】成功构建了3种穿梭质粒pFB-30-ORF5、pFB-30-ORF6和pFB-30-ORF5-OPF6,酶切鉴定正确。通过蓝白斑筛选及PCR验证后获得重组杆粒,分别命名为Bacmind-30-ORF5、Bacmind-30-ORF6和Bacmind-30-ORF5-ORF6。重组杆粒感染SF9细胞120h时出现明显的细胞病变,收获病毒液后,在透射电子显微镜可观察到大小为50nm左右呈现球形结构的VLPs。免疫电镜可以观察到胶体金颗粒结合在VLPs周围;IFA结果显示实验组均出现了明显绿色的特异性荧光灶;Western blotting结果表明,3种VLPs均出现特异性条带,并与预期大小一致。【结论】制备了3种NADC30-like谱系PRRSV的病毒样颗粒,为针对PRRSV新谱系流行株疫苗的研发奠定了基础。     

Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana

Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. ‘Pei chiao’ (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium‐mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%–0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2‐week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination‐dependent gradational increase in the elicitation of serum and saliva anti‐PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV.     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK⁻/gE⁻/GP5⁺ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK⁻/gE⁻/LacZ⁺, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK⁻/gE⁻/LacZ⁺, resulting in a PRV recombinant named TK⁻/gE⁻/GP5m⁺, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK⁻/gE⁻/GP5m⁺ and TK⁻/gE⁻/GP5⁺ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK⁻/gE⁻/GP5m⁺ against PRRSV were higher than that induced by TK⁻/gE⁻/GP5⁺. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV. Translated from Journal of Biotechnology, 2005, 21(6): 858–864 [译自: 生物工程学报]     

猪繁殖与呼吸综合征病毒非结构蛋白NSP2的原核表达及其单克隆抗体的研制

将猪繁殖与呼吸综合征病毒(PRRSV)S1株NSP2蛋白基因进行截短修饰 (tNSP2),克隆于pGEX-6P-1载体,转化大肠杆菌后用IPTG诱导表达。Western-blot结果表明,融合表达的GST-tNSP2蛋白能被PRRSV阳性血清特异性识别,大小约50kD。经GST柱提取纯化蛋白GST- tNSP2,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞进行融合,获得2株能稳定分泌抗NSP2蛋白抗体的杂交瘤细胞株,将其命名为2B5、3H3。亚型鉴定结果均为IgG 1型,其轻链均为Κ链。间接免疫荧光试验证明,2B5和3H3均能与PRRSV S1毒株产生特异性反应,而不能与SY0608毒株反应。从而为PRRSV分离株的鉴定及NSP2功能研究奠定了重要基础。     

Genetic analysis of ORF5 in porcine reproductive and respiratory syndrome virus in Japan

In recent years, no reports regarding genetic information on porcine reproductive and respiratory syndrome virus (PRRSV) with a focus on Japan have been published. To clarify the effect of time on PRRSV genomic evolution, we sequenced the open reading frame 5 (600 or 603 bases) obtained from Japanese PRRSV isolates for three periods (1992-1993, 2000-2001, and 2007-2008) and compared their phylogenetic relationships. Assessment of mean pairwise homology of nucleotide sequences of PRRSV isolates indicated a trend towards increasing heterogeneity over time. In addition, we newly detected a virus classified in cluster IV, indicative of the increasing genetic variation of PRRSV in Japan.