摘要
猪流行性腹泻(porcine epidemic diarrhea, PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)引起的一种猪的急性、高度接触性肠道传染病。自1978年报道以来在欧洲以及亚洲许多国家相继爆发,给养猪业带来巨大损失,因此对于PED的诊断以及疫苗的研究就显得尤为重要。PEDV纤突糖蛋白(S)是位于病毒粒子表面的结构蛋白,它既含有介导病毒侵入宿主细胞的受体结合域,又拥有介导机体产生病毒中和抗体的抗原表位,具有良好的免疫原性。
利用生物学软件对PEDV S基因进行了抗原位点分析,在不破坏线性抗原位点的前提下将S基因进行分割获得S基因的片段命名为S1。本试验在高效表达PEDV S1蛋白后,利用噬菌体展示技术筛选并鉴定了能与PEDV S1蛋白相互作用的多肽分子,为进行PED的快速诊断以及开发有效的新型疫苗奠定了基础。
本试验成功构建了编码PEDV S1蛋白的pET30a-S1原核重组表达质粒,并在E.Coli Rosetta中进行原核表达。将制备并纯化的重组蛋白进行复性和活性检测,结果表明这种融合蛋白具有一定的生物学活性。以重组蛋白为免疫原免疫新西兰白兔制备了多克隆抗体。免疫荧光实验以及病毒抑制实验证明此多克隆抗体具有良好的生物学活性。
利用噬菌体随机十二肽库,以PEDV S1重组蛋白为靶分子,对其进行了四轮生物淘选。对第四轮洗脱物随机淘选出的10个噬菌体单克隆的核苷酸序列进行测定,并进行多肽序列的推导,结果表明,经过对靶分子的四轮筛选后,最终筛选出三个具有高度同源性的多肽,序列分别是MPAVMSSAQVPR,NLSNRLNLSPGI,YVIHQPYAMALR。ELISA方法对合成多肽的检测结果表明这三条多肽均能与重组的PEDV S1蛋白结合。病毒体外抑制实验表明所筛选出来的其中两条多肽M和N具有良好的生物活性。体内实验通过免疫昆明鼠检测了多肽的抗病毒活性,结果表明多肽M、N在小鼠体内起到了一定的中和病毒的作用。本实验为建立新的PED诊断方法以及新型多肽疫苗的开发提供了一定的理论基础和实验依据。
Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV). Since 1978, the outbreak of PED in Europe and many Asian countries have led to huge economic losses in pig industry. Thus, there is a urgent need to understand its pathological mechanism and to develop novel PED vaccine for the prevention of PED.The S protein of PEDV is a glycoprotein localized on the virion surface, which plays a key role in the specific receptor binding, cell membrane fusion, and induction of neutralizing antibody for its possessing neutralizing epitopes and receptor binding domain. Given this, screening and identification of receptor binding domain and antigenic epitopes were carried out in this study. It will provide valuable information for development of diagnosis technology, novel vaccine, and designing antiviral immunization strategy of PED.
The experiment successfully constructed a recombinant expression vector pET-30a-S1. High level expression of S1 protein was analyzed by SDS-PAGE at different time at 37℃after IPTG induction. The S1 protein was purified and injected to a rabbit to generate polyclonal antibody. Western-blotting result showed polyclonal antibody could specifically bind to PEDV S1 protein, which indicated that recombinant fusion protein has excellent immunogenicity.
Random 12-mer Phage Display Peptide Library was used to panning the S1 protein for four rounds. Ten monoclonal phages were selected randomly and subjected to sequence comparison. The results showed that three different peptides were screened by panning on the S1 recombinat protein. Three peptides that have the highest binding activity to S1 were synthesized. Their sequences are MPAVMSSAQVPR named M, NLSNRLNLSPGI named N and YVIHQPYAMALR named Y, respectively. ELISA showed that both peptides could bind to S1 protein and inhibit cell infection by PEDV in vitro. The immunized Kunming mice were used to detect the anti-viral activity of peptides in vivo. The results showed that the peptide M and N could neutralize viral infection activity. The current study should be helpful for establishing specific diagnosis method and developing new subunit vaccine for PEDV.
引文
蔡宝祥. 1982.介绍几种新近发现的猪传染病.畜牧与兽医. (5): 218~221.
蔡炯,钟彦伟. 2010.噬菌体展示技术筛选甲型H1N1流感病毒抗原模拟表位的研究.解放军医学杂志. 61~63.
陈茹,罗琼,李树根等. 1997.间接法Dot-ELI SA检测猪流行性腹泻抗体的研究.中国兽医杂志. 23(8): 11~13.
陈新华. 2002.噬菌体随机肽库的应用进展.国外医学寄生虫病分册. 30(2): 51~54.
程庆华. 1992.青海地区猪流行性腹泻病调查青.海畜牧兽医杂志. 3(22):22~23.
崔现兰,马思奇,王明等. 1990.应用免疫荧光法诊断猪流行性腹泻的研究.中国畜禽传染病. 5: 20~24.
高慎阳,邹运明,李一经. 2007.猪流行性腹泻病毒M基因及其片段原核表达效果分析.中国预防兽医学报. 29(1): 28~30.
胡雪梅,张兆松,吴海玮等. 2002.用噬菌体肽库筛选重组日本血吸虫线粒体相关蛋白的表位.中华微生物学和免疫学杂志, 180~184.
康晓平,杨保安,赵慧等. 2005.从噬菌体库中筛选抗SARS病毒S蛋白的Fab中和活性抗体研究.中华微生物学和免疫学杂志, 634~637.
李树根,李施力,霍燕琼等. 1991.使用细胞培养单层分离猪流行性腹泻病毒闭.中国兽医科技. 10: 24~25.
林志雄,黄引贤,李树根等. 1997.应用直接免疫荧光法检测猪流行性腹泻病毒的研究.中国进出境动植检验. 2: 32~34.
刘邓. 2009.猪传染性胃肠炎和猪流行性腹泻快速诊断方法的建立及S基因的表达.硕士论文. 5~6.
刘蓬勃. 2000.抗体依赖的病毒感染增强作用.国外医学流行病学传染病学分册. 27(l): 29~32.
倪艳秀,林继煌,何孔旺,还红华. 2001.猪流行性腹泻研究概况.畜牧与兽医. 33(1): 38~40.
欧阳理,王庆林,曾宪芳等. 2002.从随机多肽库中筛选日本血吸虫抗原模拟表位诱导保护性免疫的研究.中国热带医学, 132~135.
裴敦国,葛俊伟,马广鹏等. 2008.抗猪流行性腹泻病毒N蛋白单克隆抗体制备及部分特性鉴定.东北农业大学学报. 39(7): 84~89.
钱刚,冯力,刘胜旺等. 2005.猪传染性胃肠炎病毒纤突蛋白基因的修饰及原核表达的研究.东北农业大学学报. 36(5): 611~614.
钱永清,闻人楚等, 1999.猪流行性腹泻病毒的分离培养鉴定.上海农业学报. 1(2): 41~44.
乔宪风,熊忠良,华文君等. 2001. PEDV膜蛋自基因RT-PCR最适反应条件的确立.湖北畜牧兽医. (l): 8~10.
任晓峰,李一经,李广兴等. 2002.猪传染性胃肠炎病毒S基因杆状病毒/昆虫细胞表达系统重组质粒的构建.东北农业大学学报. 33(4): 359~363.
萨姆布鲁克J,拉塞尔D W.分子克隆实验指南. 3版.黄培堂,王嘉玺,朱厚础等译.北京:科学出版社. 2002.
沈顺新,陈晓清,程庆潮等. 1996.猪流行性腹泻高免血清的制备及防治试验.中国畜禽传染病. 2: 53~54.
石娜,李广兴,任晓峰等. 2010.猪IL-15与PRRS病毒GP5基因核酸疫苗免疫效力.东北农业大学学报. 41(1): 97~102.
斯特劳B E.猪病学. 8版.赵德明,张仲秋,沈建忠等译.北京:中国农业大学出版社. 2000 : 181~187.
孙东波,冯力,时洪艳等. 2006.猪传染性胃肠炎病毒N基因的原核表达及重组蛋白的免疫活性分析.病毒学报. 22(2): 147~149.
佟有恩,冯力,李伟等. 1998.猪流行性腹泻弱毒株的培育团.中国畜禽传染病. 20(6): 329~332.
王冰,柯丽华,江红等. 1998.从随机噬菌体肽库中筛选抗草鱼出血病病毒多肽的研究. 中国病毒学. 13(4): 351~357.
王桂云等. 2001.噬菌体展示外源多肽的免疫原性研究.东北师大学报自然科学版. 101~104.
王明,马思奇,周金法等. 1993.猪流行性腹泻灭活疫苗研究明.中国畜禽传染病. 5: 17~19.
王明翠,任晓峰,李广兴. 2010.缺失信号肽和跨膜区的PRRSV GP5融合蛋白基因的表达.中国兽医科学. 40 (04): 342~346.
王明翠. 2010猪繁殖与呼吸综合征病毒GP5蛋白亲和肽筛选与鉴定.硕士论文. 8~9.
吴健敏,任兆钧,余兴龙等. 2003.猪瘟病毒EZ蛋白抗原多肽与T4噬菌体SOC蛋白的融合表达.中国兽医学报. 23(1): 14~17.
宣华,邢德坤,王殿派等. 1984.应用猪胎肠单层细胞培养猪流行性腹泻的研究.中国人民解放军兽医大学学报. 4(3): 202~207.
杨乔欣等. 2001.递呈单纯疱疹病毒糖蛋白C模拟短肽的噬菌体免疫效果的初步研究.中国人兽共患病杂志. 10~12.
杨清浩,王祥卫. 2005.噬菌体随机肽库筛选MUC1抗原模拟表位.第三军医大学学报. 1332~1334.
殷震,刘景华. 1997.动物病毒学.第二版.北京:科学出版社.
于强. 1987.应用ELISA间接法检测猪流行性腹泻抗体的研究.兽医大学学报. 2: 149~154.
余丽芸,侯喜林. 2005.流行性腹泻病毒M基因与甲病毒载体重组RNA的构建.动物医学进展. 26(5): 73~76.
张桂红. 2008.猪流行性腹泻病毒地方毒株LJB/03株分离鉴定.硕士论文. 2~3.
张强敏,郭福生,尹燕博等. 2002.猪流行性腹泻病毒分子生物学特征.中国病毒学. 17 (4): 381~384.
张晓光,韩炯,药立波等. 2002.噬菌体展示肽文库及其应用.生命科学. 14(2): 122~124.
朱维正,王新平,常建兵. 1988.应用ELISA(双抗体夹心法)检测猪流行性腹泻病猪粪便中的病毒抗原.兽医大学学报. 2: 127~129.
朱维正,王新平,常建兵. 1988.应用ELISA(双抗体夹心法)检测猪流行性腹泻病猪粪便中的病毒抗原.兽医大学学报. 2: 127~129.
朱维正,郑瑞峰. 1990.猪流行性腹泻血清学诊断法的研究.中国畜禽传染病. (1): 16~19.
Ara P W., Kolonin M G., TrePel M., et al. 2002. Steps toward mapping the human vasculature by Phage display. Nat Med. 8: 121~7.
ASA I T. 2005. OKU NI Liposomalized oligo peptides in cancer therapy. Methods Enzymol. 391: 1632~1761.
Baba T W., Liska V., Hofmann-Lehmann R., et al. 2000. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med. 6: 200~206.
Babcock G J., Esshaki D J., Thomas W D., et al. 2004. Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J Virol. 78(9): 4552~4560.
Ballesteros M L., Sánchez C M., En juanes L. 1997. Two amino acid changes at the N-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism. Virology. 227(2): 378~388.
Beate S., Hans J G., Reinhard B., et al. 1991. The S protein of bovine coronavirus is a hemagglutinin recognizing 9-0-acetylated sialic acid as a receptor determinant. J Virol. 65(11): 6232~6237.
Beckmann C., Haase B., Timmis K N., et al. 1998. Multifunctional g3p-peptide tag for current phage display systems. Journal of immunological methods. 212(2): 131~138.
Benfield D A., Nelson E., Collins J E., et al. 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolateATCCVR-2332) . J. Vet. 4: 127~133.
Bernasconi C., Guscetti F., Utiger A.,et al. 1995. Experimental infection of gnotobiotic piglets with a cell culture adapted porcine epidemic diarrhoea virus: clinical, histopathological and immunohistochemical findings.Proceedings of the 3rd ESVV Congress. Interlaken. Switzerland. 542~546.
BOEHM T., FOLKMAN J., BROWDERT., et al. 1997. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. I Nature. 390(6658): 4042~4071.
Bonavia A., Zelus B D., Wentworth D.E.,et al. 2003. Identification of a Receptor-Binding Domain of the Spike Glycoprotein of Human Coronavirus HCoV-229E. J Virol. 77(4): 2530~2538.
Bos E C., Luytjes W., van der Meulen H V., et al. 1996. The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology. 218 (1): 52~60.
Brian D A., Baric R S. 2005. Coronavirus genome structure and replication.Curr Top Microbiol Immunol. 287: 1~30.
Bridgen A, Duarte M, Tobler K, et al. 1993. Sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus. J Gen Virol. 74: 1795~1804.
Bridgen A., Duarte M., Tobler K.,et al. 1993. Ackermann M Sequence determination of thenucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus. J Gen Virol. 74(9): 1795~1804.
Bridgen A., kocherhans R., Tobler K., et al. 1998. Further Analysis of the Genome of Porcine Epidemic Diarrhoea Virus. Sdv Exp Med Biol. 440: 781~786.
Bridgen A., Kocherhans R., Tobler K., et al. 1998. Further analysis of the genome of porcine epidemic diarrhoea virus. Adv Exp Med Biol. 440: 781~786.
Cancel-Tirado S M., Yoon K J., et al. 2003. Antibody dependent enhancement of virus infection and disease. Viral Immunol. 16: 69~86.
Carvajal A., Diego R., Lanza I., et al. 1995b. Evaluation of a blocking ELISA using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies. J Vet Diagn Invest. 7(1): 60~64.
Carvajal A., Diego R., Lanza I., et al. 1995c. Seroprevalence of porcine epidemic diarrhea virus infection among different types of breeding swine farms in Spain. Preventive Vet Med. 23(1): 33~40.
Chae C., Kim O., Choi C., et al. 2000. Prevalence of porcine epidemic diarrhea virus and transmissible gastroenteritis virus infection in Korean pigs. Vet Rec. 147(21): 606~608.
Chasey D., Cartwright S.F. 1978. Virus-like particles associated with porcine epidemic diarrhea. Res Vet Sci. 25(2): 255~256.
Cheng Q H., Niu X Y. 1992. Investigation on epidemic diarrhea in pigs in Qinghai region. Chinese Qinghai Journal of Animal and Veterinary Science. 22(3): 22~23.
Cruz D J., Kim C J., Shin H J. 2006. Phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus(PEDV)neutralizing epitopes. Virology. 354(1): 28~34.
Cruz D J., Shin H J. 2007. Application of a focus formation assay for detection and titration of porcine epidemic diarrhea virus. J Virol Methods. 145(1): 56~61.
Cruz DJ., Kim CJ., Shin HJ. 2008. The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus. Virus Res. 132(1-2): 192~196.
De Arriba M L., Carvajal A., Pozo J., et al. 2002. Isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and antibody responses in serum of conventional pigs inoculated with PEDV. Vet Immunol Immunopathol. 84(1-2): 1~16.
De Arriba M L., Carvajal A., Pozo J., et al. 2002. Lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus. J Virol Methods. 105(1): 37~47.
Debouck P., Pensaert M. 1980. Experimental infection of pigs with a new porcine enteric coronavirus, CV 777. J Vet Res. 41(2): 219-223.
Debouck P., Pensaert M., Coussement W. 1981a. The pathogenesis of an enteric infection in pigs experimentally induced by the coronanvirus-like agent CV777. Vet microbial. 6: 157~165.
Duarte M., Laude H. 1994. Sequence of the spike protein of the porcine epidemic diarrhoea virus. J Gen Virol. 75: 1195~1200.
Duarte M., Laude H. 1994. Sequence of the spike protein of the porcine epidemic diarrhea virus. Gen Virol. 75: 1195~1200.
Duarte M., Tobler K., Bridgen A., et al. 1994. Sequence Analysis of the Porcine Epidemic Diarrhea Virus Genome between the Nucleocapsid and Spike Protein Genes Reveals a Polymorphic ORF. Virology. 198: 466~476.
Duarte M., Tobler K., Bridgen A., et al. 1994. Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF. Virology. 198(2): 466~476.
Ducatelle R., Coussement W., Pensaert M B., et al. 1981. In vivo morphogenesis of a new porcine enteric coronavirus, CV777. Arch Virol. 68: 35~44.
Guscetti F., Bernasconi C., Tobler K., et al. 1998. Immunohistochemical detection of porcine epidemic diarrhea virus compared to other methods. Clin Diagn Lab Immunol. 5(3): 412~414.
Hammond S A., Cook S J., Lichtenstein D L., et al. 1997..Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process. J. Virol. 71: 3840~3852.
HAV IV Y S., LACKWELL J L., ANERVA A., et al. 2002. Adenoviral gene therapy for renal cancer requires retargeting to alternative cellular receptors. Cancer Res. 62(15): 42732~42811.
Hofmann M., Wyler R. 1988. Propagation of the virus of PED in cell culture. J Clin Microbiol. 26: 2235~2239.
Hofmann M., Wyler R. 1989. Quantitation, Biological and Physicochemical Properties of Cell Culture-adapted Porcine Epidemic Diarrhea Coronavirus(PEDV). Vet Microbiol. 20: 131~142.
Horvath L., Moscari E. 1981.Ultrastructural changes in the small intestinal epithelium of suckling pigs affected with a transmissible gastroenteritis (TGE)-like disease. Arch Virol. 68: 103~113.
Hou X., Yu L Y., Liu J. 2007. Development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea(PEDV) antibodies. Vet Microbiol. 123(1-3): 86~92.
Hsiao K C., Brissette R E., Wang P., et al. 2003. Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2[J ]. Proteome Sci . 1(1): 1210.
HUSTON J S., MC CARTNEY J., TAI M S., et al.1993. Medical application of single-chain antibodies. Int Rev Immunol. 10(2/3): 195~217.
Ishikawa K., Sekiguchi H., Ogino T., et al. 1997. Direct and Rapid Detection of Porcine Epidemic Diarrhoea Virus by RT-PCR. J Virol Method. 69: 191~195.
Jung K., Kim J., Kim O., et al. 2003. Differentiation between Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus in Formalin-fixed Paraffin-embedded Tissues by Multiplex RT Nested PCR and Comparison with in situ Hybridization. J Virol Methods. 108(1): 41~47.
Jung-Eun Park., Deu John M., Cruz Hyun-Jin Shin. 2010. Trypsin-induced hemagglutination activity of porcine epidemic diarrhea virus. Arch Virol. 155: 595~599.
Kadoi K., Sugioka H., Satoh T., et al. 2002. The propagation of a porcine epidemic diarrheavirus in swine cell lines. New Microbiol. 25(3): 285~290.
Kal ie E., Jaitin D A., Abramovich R., et al. 2007. An IFN alpha 2 mutantoptimized by phage display for IFNAR1 binding confers specifically enhanced antitum or activities. J Biol Chem. 282(15): 602~611.
Kim O., Chae C. 2002. Comparison of Reverse Transcription Polymerase Chain Reaction Immunohistochemistry and in situ Hybridization for the Detection of Porcine Epidemic Diarrhea Virus in Pigs. J Vet Res. 66(2): 112~116.
Knuchel M., Ackermann M., Miiller H K., et al. 1992. An ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV) based on the specific solubility of the viral surface glycoprotein .Vet Microbiol. 32(2): 117~134.
Kocherhans, R., Bridgen, A., Ackermann, M., et al. 2001. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes. 23(2): 137~144.
Kubota S., Sasaki O., Amimito K., et al. 1999. Detection of Porcine Epidemic Diarrhea Virus Using Polymerase Chain Reaction and Comparison of the Nucleocapsid Protein Genes among Strains of the Virus. J Vet Med Sci. 61: 827~830.
Kusanaqi K., Kuwahara H., Katoh T., et al. 1992. Isolation and serial propagation of Porcine Epidemic Diarrhoea Virus in Cell Cultures and Partial Characterization of the Isolate.J. Vet Med. 54(2): 313~318.
Kweon C H., Lee Jq Han Mq Kang Y B. 1997. Rapid Diagnosis of Porcine Epidemic Diarrhoea Virus Infection by Polymerise Chain Reaction. J Vet Med Sci. 59(3): 231~232.
L Baoxian., G Junwei., L Yijing. 2007. Porcine Aminopeptidase N is a Functional Receptor for the PEDV Coronavirus. Virology. 5: 1~7.
Lai M M., Cavanagh D. 1997. The molecular biology of coronaviruses. Virus Res. 48: 1~100. Lee H M., Lee B J., Tae J H., et al. 2000. Detection of porcine epidemic diarrhea virus by immunohistochemistry with recombinant antibody produced in phages. J Vet Med. 62(3): 333~337.
Lili huang., Daniel J..2004. Novel peptide inhibitors of Angiotensin-converting. Enzyme Biol Chem. 279(6): 4604~4611.
Liu D X., Cavanagh D., Green P., et al. 1991. A polycistronic mRNA specified by the coronavirus infectious bronchitis virus. Virology. 184: 531~544.
Lontok E., Corse E. 2004. Machamer localization of coronavirus spike proteins C E. Intracellular targeting signals contribute to near the virus assembly site. J Viro1. 78(11): 5913~5922.
M. Duarte., H. Laude. 1994. Sequence of the spike protein of the porcine epidemic diarrhoea virus. Gen Virol. 75: 1195~1200.
Macneughton M R., Davies H A. 1978. Ribonucleoprotein-like structures from coronavirus particles. J Gen Virol. 39 (3):545-549
Mahender Singh. 1999. A Novel Internal Open Reading Frame Product Expressed from a Polycistronic Mrna of Porcine Epidemic Diarrhoea Virus May not Contribute to Virus. J Gen Virol. 80: 1959~1963.
Manoutcharian K., Gevorkian G., Cano A., et al. 2001. phage displayed biomolecules aspreventive and therapeutic agents. CurrPharmBiotechnolog. 2(3): 217~223.
Mingcui Wang., Ren X., Guangxing Li., et al. 2009. Phylogenetic characterization of genes encoding for glycoprotein 5 and membrane protein of PRRSV isolate HH08. J Vet. 10(4): 309~315.
Murhy F A., Fauquet C M. 1995. Smith Report of the ICTV. Archives virology. 10: 407~409.
Oh J S., Song D S., Yang J S., et al. 2005. Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection. J Vet. 6(4): 349~352.
Park S J., Moon H J., Luo Y.,et al. 2007. Cloning and further sequence analysis of the ORF3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses. Virus Genes. [Epub ahead of print].
Park S J., Song D S., Ha G W., et al. 2007. Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13. Virus Genes. 35(1): 55~64.
Pensaert M B., Debouck P. 1978. A new coronavirus- like particle associated with diarrhea in swine. Arch Virol. 58(3): 243-247.
Pillutla R C., Hsiao K C., Beasley J R., et al. 2002. Peptides identify the critical hot spot s involved in the biological action of the in sulin receptor. J Biol Chem. 277 (25): 22590~222594.
Pospischil A., Hess R G., Bachmann P A. 1981. Light microscopy and ultrahistology of intestinal changes in pigs infected with enzootic diarrhea virus (EVD): Comparison with transmissible gastroenteritis (TGE) virus and porcine rotavirus infections. Zentrabl Veterinarmed (B). 28:564~577.
Ren X F., LI Y J., LI G X., et al. 2002. Cloning and identification of S gene from Chinese isolate TH-98 of transmissible gastroenteritis virus. Journal of Northeast Agricultural University. 9(1): 49~54.
Ren X., Suo S., Jang Y S. 2010. Development of a porcine epidemic diarrhea virus M protein-based ELISA for virus detection[E]. Biotechnol Lett. DOI 10.1007/s10529-010-0420-8.
Rodak L., Valicek L., Smid B., et al. 2005. An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces. Vet Microbiol. 105(1): 9~17.
Rosenfeld S A., Nadeau D., Tirado J., et al. 1996. Production and Purification of recombinant hirudin Expressed in the methylotroPhic yeast Piehia pastoris. Protein Expr purif. 8(4): 476~482.
Rottier P J M., Welling G W., Welling-Wester S., et al. 1986. Predicted membrane topology of the coronavirus protein E1. Proc Natl Acad. 81: 1421~1425.
Schmitz A., Tober K., Suter M., et al. 1998. Prokaryotic Expression of Porcine Epidemic Diarrhoea ORF3. Adv Exp Med Biol. 440: 775~780.
Schwegmann-Wessels C., Herrler G. 2006. Transmissible gastroenteritis virus infection: a vanishing specter. Dtsch Tierarztl Wochenschr. 113: 157~159.
Sidhu., Sachdev S. 2001. Engineering M13 for phage display. Biomolecular Engineering. 18(2): 57~63.
Smith G P. 1985. Filamentous fusion phage: novel expression vecyers the display cloned antigens on the virion surface. Science. 288(4705): 1315~1317.
Snijder, E J., Meulenberg, J M., et al. 2001. Arterivirusesin Fields Virology. 1(4): 1205~1220.
Song D S., Kang B K., Lee S S., et al. 2006. Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs. J Virol Methods. 133(1): 27~33.
Spaan W., Cavanagh D., et al. 1988. Coronavirus: structure and genome expression. Gen. Virol. 69: 29~52.
Subramanian K N., Weisman L E., Rhodes T., et al. 1998. Safety, tolerance and pharmacokinetics of a humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. Pediatr. Infect. 17: 110~115.
Sueyoshi M., Tsuda T., Yamazaki K.,et al. 1995. An immunohistochemical investigation of porcine epidemicdiarrhea. J Comp Pathol. 113(1): 59~67.
Sun D B., Feng L., Shi H Y., et al. 2007. Spike protein region of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies. Acta Virol. 51(3): 149~56.
Sun D., Feng L., Shi H., et al. 2008. Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein. Vet Microbiol. 131(1-2): 73~81.
Sun-Hwa Chang., Jong-Lye Bae., Tae-Jin Kang., et al. 2002. Identification of the Epitope Region Capable of Inducing Neutralizing Antibodies against the Porcine Epidemic Diarrhea Virus. Mol Cells Vol. 14(2): 295~299.
T J Kang., J E Seo., D H Kim., et al. 2003. Analysis of the Korean strain of spike gene of porcine epidemic diarrhea virus and expression. Cloning and Sequence Analysis of the SG Yeo, M Hernandez, PJ Krell,ééNagy. Spike Gene of Porcine Epidemic Diarrhea Virus Chinju99. Virus Genes. 41(2): 378~83.
T J Kang., K H Kang., J A Kim.,et al. 2004. High-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco. Protein Expr Purif. 38(1): 129~35.
Takahashi K., Okada K., Ohshima K. 1983. An outbreak of swine diarrhea of a new-type associated with Coronavirus-like Particles in Japan. Vet Sci. 45: 829~832.
Tang Z., Yu Y., Zhou X., et al. 1997. Three decades′experience in surgery of hepatocellular carinoma. Chin Med J. 110: 245~9.
Timani K A., Liao Q J., Ye L B., et al. 2005. Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirus. Virus Res. 114: 23~34.
Tobler K., Ackermann M. 1995. PEDV leader sequence and junction sites. Adv Exp Med Biol. 380: 541~542.
Utiger A., Rkosskopf M., Guscetti F., et al. 1993. Preliminary Characterization of A Monoclonal Antibody Specific for a Viral 27kD Glycoprotein Family Synthesized in Porcine Epidemic Diarrhoea Virus Infected Cells. Adv Exp Med Bio1. 342: 197~202.
Utiger A., Tobler K., Bridgen A., et al. 1995. Identification of Protein Specified by Porcine Epidemic Diarrhea Virus. Adv Exp Med Biol. 380: 287~290.
Van Reeth K., Pensaert M. 1994. Prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds. Vet Rec. 135(25): 594~597.
Wensvoort G., Terpstra C., Pol J M A., et al. 1991. Mystery swine disease in the Netherlands: the isolation of Lelystad virus. Vet. 13: 121~130.
Wu J., Ma Q N., Lam K S.1994. Identifying substrate motifs of protein kinases by a random library approach. Biochemistry. 33(49): 14825~14833.
Zimmerman J., Sanderson T., Eernisse K., et al. 1992. Transmission of SIRS virus in convalescent animals to commingled penmates under experimental conditions. Swine Pract. Newslett. 4: 25.