PRRSV GP5蛋白与泛素融合蛋白基因重组质粒的构建与免疫特性研究
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摘要
猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)是目前危害养猪业重要的病原之一。GP5蛋白是该病毒的主要保护性抗原,含有中和性抗原表位,但猪感染PRRSV后产生中和抗体的时间较晚,而且其滴度较低。泛素(Ubiquitin,简称Ub)是一个由76个氨基酸残基组成的非常保守的小蛋白质。泛素依赖性的蛋白质降解途径是目前已知的重要的、有高度选择性的蛋白质降解途径之一。Ub可用于增强DNA疫苗免疫反应。我们假设PRRSV保护性免疫力较低可能与糖基化蛋白中和抗原表位空间结构有关,所以,本研究将Ub基因插入GP5基因5端,旨在有效的将糖基化蛋白分解成小的多肽,促进不同抗原表位提呈,增强免疫效果,为PRRSV DNA疫苗提供理论依据。
根据GenBank数据库中的PRRSV S1毒株GP5基因序列设计合成了两对分别含有Bg/Ⅱ、NotI酶切位点的引物,以pcDNA3-ORF5质粒为模板用PCR技术获得大小为620bp左右的目的产物,经纯化后与真核表达载体pCMV-Ub同时进行酶切,分别经回收后连接。连接产物转化DH5α感受态细菌,然后在氨苄青霉素抗性(Amp~R)平板上挑取阳性菌落,过夜培养,提取质粒,经单酶切、双酶切鉴定及DNA序列测定。用DNAStar软件分析结果表明:扩增片段与预期片段大小相符,且完全符合载体阅读框。该毒株GP5基因序列与美洲标准毒株和其它分离株的基因同源性为99%。最后分别命名为pCMV-Ub-GP5、pCMV-GP5。
用PEG2000提取纯化了重组质粒pCMV-GP5和pCMV-Ub-GP5,将重组质粒组以100μg背部皮内注射免疫BALB/c小鼠,以间隔15天的频率共免疫4次,用中和抗体试验和ELISA试验测定不同时间小鼠血清PRRSV抗体,以比较GP5基因和与泛素融合表达GP5基因的免疫特性。结果表明,第三次免疫后14天(即首免后44天)pcMV-ub-GP5和pcMV-GP5免疫组的小鼠血清ELISA试验OD_(490)值均能达到1.0,且均可检测到中和抗体的存在,在第4次免疫后14d(即首免后59d)中和抗体平均涌厦为7和12,抗体产生时间和抗体滴度差异不显著。
以上研究结果表明,本文成功构建了PRRSV GP5蛋白和泛素融合蛋白基因重组质粒pCMV-GP5和pCMV-Ub-GP5。重组质粒DNA免疫小鼠都能诱导产生抗PRRSV的特异性体液免疫应答,两者无显著差异,本试验泛素化的GP5的细胞免疫特性有待进一
Porcine reproductive and respiratory syndrome virus(PRRSV), a causative agent of highly contagious disease with worldwide distribution, can cause severe economic losses to the swine industry. The GP5 is one of the major factors of the virus to which the host responses well with protective antibody production. The recombinant plasmids containing the GP5 gene can induce specific immune responses against PRRSV. The ubiquitin-proteasome pathway is an efficient pathway for protein degradation. It's mainly responsible for the selective degradation of many protins in eukaryotic cells.The Ub fusion vaccines were designed to accelerate turnover of transgene antigen and increase the variety and number of peptides available for major histocompatibility complex (MHC) binding.Two pairs of specific primers which have the restriction enzyme site BglⅡ、 NotⅠ respectively were designed. About 620bp PCR product was obtained from pcDNA3-ORF5 plasmid that had been constructed with PCR. The product of PCR and pCMV-Ub vector were digested by the restriction enzymes, and purified, respectively, and ligased. The ligation was transformed into E.coli strain DH5 a competent cells and selected with ampicillin. Positive clones were picked up on the LB plate with ampicillin after 12h and then inoculated into LB culture. A small scale of plasmid was extracted and identified by restriction enzymes and sequencing. The results showed that the GP5 gene was cloned into pCMV in the correct open reading frame, and they were named pCMV-Ub-GP5 and pCMV-GP5 respectively. The nucleotide homogeneity between GP5 of S1 strain and other ioalated strain was analyzed in the NCBI with Blast biosoft, and the homogeneity was about 99%.The efficacy of PRRSV GP5 DNA vaccines between pCMV-Ub-GP5 and pCMV-GP5 were compared. Mice in each group were immunized with 100μg of plasmid by
intra-dermal injection. All groups were immunized four times in all with frequency of 15 days. The results showed that the humoral responses were both induced in group pCMV-Ub-GP5 and pCMV-GP5. The level of ELISA antibody and neutralizing antibody was equivalent between group pCMV-Ub-GP5 and group pCMV-GP5. On time of 15d after the third immunization, the OD490 of ELISA antibody of both groups reached 1.0 and neutralizing antibody could be detected. On time of 15d after the fourth immunization, neutralizing antibody of group CMV-Ub-GP5 reached 7 and group CMV-GP5 reached 12.In conclusion, the present study demonstrated that the recombinant plasmid of ubiquitin-conjugated GP5 were constructed and the plasmid induced humoral response in mice, as well as the recombinant plasmid with GP5 gene only. The cellular immunity of recombinant plasmids should be studied furtherly.
根据GenBank数据库中的PRRSV S1毒株GP5基因序列设计合成了两对分别含有Bg/Ⅱ、NotI酶切位点的引物,以pcDNA3-ORF5质粒为模板用PCR技术获得大小为620bp左右的目的产物,经纯化后与真核表达载体pCMV-Ub同时进行酶切,分别经回收后连接。连接产物转化DH5α感受态细菌,然后在氨苄青霉素抗性(Amp~R)平板上挑取阳性菌落,过夜培养,提取质粒,经单酶切、双酶切鉴定及DNA序列测定。用DNAStar软件分析结果表明:扩增片段与预期片段大小相符,且完全符合载体阅读框。该毒株GP5基因序列与美洲标准毒株和其它分离株的基因同源性为99%。最后分别命名为pCMV-Ub-GP5、pCMV-GP5。
用PEG2000提取纯化了重组质粒pCMV-GP5和pCMV-Ub-GP5,将重组质粒组以100μg背部皮内注射免疫BALB/c小鼠,以间隔15天的频率共免疫4次,用中和抗体试验和ELISA试验测定不同时间小鼠血清PRRSV抗体,以比较GP5基因和与泛素融合表达GP5基因的免疫特性。结果表明,第三次免疫后14天(即首免后44天)pcMV-ub-GP5和pcMV-GP5免疫组的小鼠血清ELISA试验OD_(490)值均能达到1.0,且均可检测到中和抗体的存在,在第4次免疫后14d(即首免后59d)中和抗体平均涌厦为7和12,抗体产生时间和抗体滴度差异不显著。
以上研究结果表明,本文成功构建了PRRSV GP5蛋白和泛素融合蛋白基因重组质粒pCMV-GP5和pCMV-Ub-GP5。重组质粒DNA免疫小鼠都能诱导产生抗PRRSV的特异性体液免疫应答,两者无显著差异,本试验泛素化的GP5的细胞免疫特性有待进一
Porcine reproductive and respiratory syndrome virus(PRRSV), a causative agent of highly contagious disease with worldwide distribution, can cause severe economic losses to the swine industry. The GP5 is one of the major factors of the virus to which the host responses well with protective antibody production. The recombinant plasmids containing the GP5 gene can induce specific immune responses against PRRSV. The ubiquitin-proteasome pathway is an efficient pathway for protein degradation. It's mainly responsible for the selective degradation of many protins in eukaryotic cells.The Ub fusion vaccines were designed to accelerate turnover of transgene antigen and increase the variety and number of peptides available for major histocompatibility complex (MHC) binding.Two pairs of specific primers which have the restriction enzyme site BglⅡ、 NotⅠ respectively were designed. About 620bp PCR product was obtained from pcDNA3-ORF5 plasmid that had been constructed with PCR. The product of PCR and pCMV-Ub vector were digested by the restriction enzymes, and purified, respectively, and ligased. The ligation was transformed into E.coli strain DH5 a competent cells and selected with ampicillin. Positive clones were picked up on the LB plate with ampicillin after 12h and then inoculated into LB culture. A small scale of plasmid was extracted and identified by restriction enzymes and sequencing. The results showed that the GP5 gene was cloned into pCMV in the correct open reading frame, and they were named pCMV-Ub-GP5 and pCMV-GP5 respectively. The nucleotide homogeneity between GP5 of S1 strain and other ioalated strain was analyzed in the NCBI with Blast biosoft, and the homogeneity was about 99%.The efficacy of PRRSV GP5 DNA vaccines between pCMV-Ub-GP5 and pCMV-GP5 were compared. Mice in each group were immunized with 100μg of plasmid by
intra-dermal injection. All groups were immunized four times in all with frequency of 15 days. The results showed that the humoral responses were both induced in group pCMV-Ub-GP5 and pCMV-GP5. The level of ELISA antibody and neutralizing antibody was equivalent between group pCMV-Ub-GP5 and group pCMV-GP5. On time of 15d after the third immunization, the OD490 of ELISA antibody of both groups reached 1.0 and neutralizing antibody could be detected. On time of 15d after the fourth immunization, neutralizing antibody of group CMV-Ub-GP5 reached 7 and group CMV-GP5 reached 12.In conclusion, the present study demonstrated that the recombinant plasmid of ubiquitin-conjugated GP5 were constructed and the plasmid induced humoral response in mice, as well as the recombinant plasmid with GP5 gene only. The cellular immunity of recombinant plasmids should be studied furtherly.
引文
[1] Blodeau R. Dea S, Sauvageau RA, et al. Vet. Rec. ,1991, 129:102-103.
[2] Keffaber KK. Reproductive failure of unknown etiology[J]. Am Assoc Swine Pract Newslett, 1989,1:1-9.
[3] Wensvoort G, Terpstra C, Pol JMA, et al. Mystery swine disease in the Netherlands isolation of Lelystad virus [J]. Vet Q, 1991, 13:121-130.
[4] 郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病,1996,(2):1-4.
[5] 郭宝清,陈章水,刘文兴,等.应用间接荧光法从国内生殖障碍综合征猪群中检测生殖和呼吸综合征阳性抗体的研究[J].中国兽医科技,1996,(3):3-5.
[6] Meulenberg JJ, Hulst MM, de Meijer EJ, et al. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology. 1993. 192(1): 62-72.
[7] Drew TW. Comparative serology of porcine reproductive and respiratory syndrome in eight European laboratories, using immunoperoxidase monolayer assay and enzyme-linked immunosorbent assay. Rev Sci Tech. 1995, 14(3): 761-75.
[8] Snijder E, Meulenberg J J M. The molecular biology of arteriviruses [J]. J Gen Virol, 1998, 79:961-979.
[9] Wu W H, Fang Y, Farwell R, et al. A 10kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b[J]. Virology, 2001, 287:183-191.
[10] Meng X J. Heterogeneity of porcine reproductive and respiratory syndrome virus : implications for current vaccine efficacy and future vaccine development [J]. Vet Microbiol, 2000, 74:309-329.
[11] Van Nieuwstadt AP, Meulenberg JJM, van EssenZandbergen A, et al., 1996. Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus(Ateriviridae) are structural proteins of the virion. J Virol 70:4767-4772
[12] Meulenberg J J M, Petersen-den Besten A, Kluyver E P, et al. Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus [J]. Virology, 1995, 206:155-163.
[13] Plana Duran J, Climent I, Sarraseca J, et al. Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 protins in protection. Virus Genes. 1997, 14(1): 19-29.
[14] Pirzadeh, B., and S. Dea. Immune response in pigs vaccinated with plasmid DNA encoding ORF5
??of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 1998, 79: 989-999.
[15] Weiland E, Wieczorek-Krohmer M, Kohl D, et al Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4. Vet Microbiol. 1999, 66(3): 171-86.
[16] Rowland RR, Steffen M, Ackerman T, et al. The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332. Virology. 1999, 259(2): 262-6.
[17] Ostrowski M, Galeota JA, Jar AM, et al. Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J Virol. 2002, 76(9): 4241-50.
[18] Rost B. PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 1996, 266: 525-39.
[ 19] Takikawa N, Kobayashi S, Ide S, et al. Detection of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus in swine sera by enzyme-linked immunosorbent assay. J Vet Med Sci. 1996, 58(4): 355-7.
[20] Molitor TW, Bautista EM, Choi CS. Immunity to PRRSV: double-edged sword. Vet Microbiol. 1997, 55(1-4): 265-76.
[21] Loemba. H.D., S. Mounir, H. Mardassi, et al. Kinetics of humoral immune response to the major structuralproteins of the porcine reproductive and respiratory syndrome virus. Arch. Virol. 1996, 141: 751-761.
[22] Weiland, E., M. Wieczorek-Krohmer, D. Kohl, et al. Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4. Vet. Microbiol. 1999, 66: 171-186.
[23] Pirzadeh, B., and S. Dea. Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. J. Gen. Virol. 1997, 78: 1867-1873.
[24] Joo. H.S.. B.K. Park, S.A. Dee, et al. 1997. Indirect fluorescent IgM antibody response of pigs infected with porcine reproductive and respiratory syndrome syndrome virus. Vet. Microbiol. 55:303-307.
[25] Loemba, H.D., S. Mounir, H. Mardassi, et al. 1996. Kinetics of humoral immune response to the major structuralproteins of the porcine reproductive and respiratory syndrome virus. Arch. Virol. 141:751-761.
[26] Park, B.K., H.S. Joo, S.A. Dee, et al. 1995. Evaluation of an indirect fluorescent IgM antibody test for the detection of pigs with recent infection of porcine reproductive and respiratory syndrome virus. J. Vet. Diagn. Invest. 7:544-546.
[27] Yoon, K. J., J. J. Zimmerman, S. L. Swenson, et al. 1995. Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection. J. Vet. Diagn. Invest. 7:305-312.
[28] Nelson, E. A., J. Christopher-Hennings, and D. Benfield. 1994. Serum immune response to the proteins of porcine reproductive and respiratory syndrome (PRRS) virus. J. Vet. Diagn. Invest. 6:410-415.
[29] Nielsen, J., and A. Bφther. 1997. Hematological and immunological parameters of 41/2-month old pigs infectedwith PRRS virus. Vet. Microbiol. 55:289-294.
[30] Lager, K. M., W. L. Mengeling, and S. L. Brockmeier. 1997. Duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine. Vet. Microbiol. 58:127-133.
[31] Bautista, E. M., and T. W. Molitor. Cell-mediated immunity to porcine reproductive and respiatory syndrome virus in swine. Viral Immunol. 1997, 80: 83-94.
[32] Shimizu M, Yamada S, Murakami Y, et al. Isolation of porcine reproductive and respiratory syndrome (PRRS) virus from Heko-Heko disease of pigs. J Vet Med Sci. 1994, 56(2): 389-91.
[33] Kwang, J., H. S. Kim, and H. S. Joo. Cloning, expression, and sequence analysis of the ORF4 gene of the porcine reproductive and respiratory syndrome virus MN-1b. J. Vet. Diagn. Invest, 1994, 6: 293-296.
[34] Kwang. J.. F. Zuckermann, G. Ross, et al. Antibody and cellular immune responses of swine following immunization with plasmid DNA encoding the PRRS virus ORF's 4, 5, 6 and 7. Res, Vet. Sci. 1999. 67:199-201
[35] Mardassi H, Mounir S, Dea S. Molecular analysis of the ORFs 3 to 7 of porcine reproductive and respiratory syndrome virus, Quebec reference strain. Arch Virol. 1995, 140(8): 1405-18.
[36] 仇华吉,童光志。猪生殖-呼吸综合征。吉林科学技术出版社。
[37] Nelson EA, Christopher-Hennings J, Drew T, Wensvoort G, Collins JE, Benfield DA. Differentiation of US and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. J Clin Microbiol 1993, 31:3184-9.
[38] Allende R, Lewis TL, Lu Z, Rock DL, Kutish GF, Ali A, et al. North American and European porcine reproductive and respiratory syndrome viruses differs in non-structural protein coding regions. J Gen Virol, 1999.80:307-15.
[39] Sur J H, Doster A R, Osorio F A. Apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus[J]. Vet Pathol, 1998, 35(6) : 506～514.
[40] Delputte P L ,Vanderheijden N ,Nauwynck H J ,Pensaert M B. Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages[J ]. J Virol ,2002 ,76(9) :4312 ~ 4320.
[41 ] Sur J H ,Doster A R .Osorio F A. Apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus[J ]. Vet Pathol ,1998 ,35(6): 506 -514.
[42] Weiland E , Wieczorek2Krohmer M , Kohl D , et al. Monoclonal antibodies to the E of porcine reproductive and respiratory syndrome virus are more effective in virus neut ralization than monoclonal antibodies to the GP4 [ J ]. Vet Microbiol ,1999 ,66 (3): 1712186.
[43] Bautista EM, Molitor TW. T cell response to the structural polypeptides of porcine reproductive and respiratory syndrome virus.Arch Virol 1997,144:117-1134.
[2] Keffaber KK. Reproductive failure of unknown etiology[J]. Am Assoc Swine Pract Newslett, 1989,1:1-9.
[3] Wensvoort G, Terpstra C, Pol JMA, et al. Mystery swine disease in the Netherlands isolation of Lelystad virus [J]. Vet Q, 1991, 13:121-130.
[4] 郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病,1996,(2):1-4.
[5] 郭宝清,陈章水,刘文兴,等.应用间接荧光法从国内生殖障碍综合征猪群中检测生殖和呼吸综合征阳性抗体的研究[J].中国兽医科技,1996,(3):3-5.
[6] Meulenberg JJ, Hulst MM, de Meijer EJ, et al. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology. 1993. 192(1): 62-72.
[7] Drew TW. Comparative serology of porcine reproductive and respiratory syndrome in eight European laboratories, using immunoperoxidase monolayer assay and enzyme-linked immunosorbent assay. Rev Sci Tech. 1995, 14(3): 761-75.
[8] Snijder E, Meulenberg J J M. The molecular biology of arteriviruses [J]. J Gen Virol, 1998, 79:961-979.
[9] Wu W H, Fang Y, Farwell R, et al. A 10kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b[J]. Virology, 2001, 287:183-191.
[10] Meng X J. Heterogeneity of porcine reproductive and respiratory syndrome virus : implications for current vaccine efficacy and future vaccine development [J]. Vet Microbiol, 2000, 74:309-329.
[11] Van Nieuwstadt AP, Meulenberg JJM, van EssenZandbergen A, et al., 1996. Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus(Ateriviridae) are structural proteins of the virion. J Virol 70:4767-4772
[12] Meulenberg J J M, Petersen-den Besten A, Kluyver E P, et al. Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus [J]. Virology, 1995, 206:155-163.
[13] Plana Duran J, Climent I, Sarraseca J, et al. Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 protins in protection. Virus Genes. 1997, 14(1): 19-29.
[14] Pirzadeh, B., and S. Dea. Immune response in pigs vaccinated with plasmid DNA encoding ORF5
??of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 1998, 79: 989-999.
[15] Weiland E, Wieczorek-Krohmer M, Kohl D, et al Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4. Vet Microbiol. 1999, 66(3): 171-86.
[16] Rowland RR, Steffen M, Ackerman T, et al. The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332. Virology. 1999, 259(2): 262-6.
[17] Ostrowski M, Galeota JA, Jar AM, et al. Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J Virol. 2002, 76(9): 4241-50.
[18] Rost B. PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 1996, 266: 525-39.
[ 19] Takikawa N, Kobayashi S, Ide S, et al. Detection of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus in swine sera by enzyme-linked immunosorbent assay. J Vet Med Sci. 1996, 58(4): 355-7.
[20] Molitor TW, Bautista EM, Choi CS. Immunity to PRRSV: double-edged sword. Vet Microbiol. 1997, 55(1-4): 265-76.
[21] Loemba. H.D., S. Mounir, H. Mardassi, et al. Kinetics of humoral immune response to the major structuralproteins of the porcine reproductive and respiratory syndrome virus. Arch. Virol. 1996, 141: 751-761.
[22] Weiland, E., M. Wieczorek-Krohmer, D. Kohl, et al. Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4. Vet. Microbiol. 1999, 66: 171-186.
[23] Pirzadeh, B., and S. Dea. Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. J. Gen. Virol. 1997, 78: 1867-1873.
[24] Joo. H.S.. B.K. Park, S.A. Dee, et al. 1997. Indirect fluorescent IgM antibody response of pigs infected with porcine reproductive and respiratory syndrome syndrome virus. Vet. Microbiol. 55:303-307.
[25] Loemba, H.D., S. Mounir, H. Mardassi, et al. 1996. Kinetics of humoral immune response to the major structuralproteins of the porcine reproductive and respiratory syndrome virus. Arch. Virol. 141:751-761.
[26] Park, B.K., H.S. Joo, S.A. Dee, et al. 1995. Evaluation of an indirect fluorescent IgM antibody test for the detection of pigs with recent infection of porcine reproductive and respiratory syndrome virus. J. Vet. Diagn. Invest. 7:544-546.
[27] Yoon, K. J., J. J. Zimmerman, S. L. Swenson, et al. 1995. Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection. J. Vet. Diagn. Invest. 7:305-312.
[28] Nelson, E. A., J. Christopher-Hennings, and D. Benfield. 1994. Serum immune response to the proteins of porcine reproductive and respiratory syndrome (PRRS) virus. J. Vet. Diagn. Invest. 6:410-415.
[29] Nielsen, J., and A. Bφther. 1997. Hematological and immunological parameters of 41/2-month old pigs infectedwith PRRS virus. Vet. Microbiol. 55:289-294.
[30] Lager, K. M., W. L. Mengeling, and S. L. Brockmeier. 1997. Duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine. Vet. Microbiol. 58:127-133.
[31] Bautista, E. M., and T. W. Molitor. Cell-mediated immunity to porcine reproductive and respiatory syndrome virus in swine. Viral Immunol. 1997, 80: 83-94.
[32] Shimizu M, Yamada S, Murakami Y, et al. Isolation of porcine reproductive and respiratory syndrome (PRRS) virus from Heko-Heko disease of pigs. J Vet Med Sci. 1994, 56(2): 389-91.
[33] Kwang, J., H. S. Kim, and H. S. Joo. Cloning, expression, and sequence analysis of the ORF4 gene of the porcine reproductive and respiratory syndrome virus MN-1b. J. Vet. Diagn. Invest, 1994, 6: 293-296.
[34] Kwang. J.. F. Zuckermann, G. Ross, et al. Antibody and cellular immune responses of swine following immunization with plasmid DNA encoding the PRRS virus ORF's 4, 5, 6 and 7. Res, Vet. Sci. 1999. 67:199-201
[35] Mardassi H, Mounir S, Dea S. Molecular analysis of the ORFs 3 to 7 of porcine reproductive and respiratory syndrome virus, Quebec reference strain. Arch Virol. 1995, 140(8): 1405-18.
[36] 仇华吉,童光志。猪生殖-呼吸综合征。吉林科学技术出版社。
[37] Nelson EA, Christopher-Hennings J, Drew T, Wensvoort G, Collins JE, Benfield DA. Differentiation of US and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. J Clin Microbiol 1993, 31:3184-9.
[38] Allende R, Lewis TL, Lu Z, Rock DL, Kutish GF, Ali A, et al. North American and European porcine reproductive and respiratory syndrome viruses differs in non-structural protein coding regions. J Gen Virol, 1999.80:307-15.
[39] Sur J H, Doster A R, Osorio F A. Apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus[J]. Vet Pathol, 1998, 35(6) : 506～514.
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