PEDV分离株S1基因的重组杆状病毒真核表达
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摘要
为了研究猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)分离株(PEDV/LA/2014/02)纤突蛋白(S1)的真核表达及其反应原性,本研究利用杆状病毒真核表达系统表达出重组His-PEDV-S1蛋白。利用在线软件分析PEDV S1基因在sf9细胞内的稀有密码子,经优化密码子后的基因进行人工合成,合成后的PEDV S1基因被克隆至杆状病毒的穿梭载体(pFastBac HT A)中,转化大肠杆菌DH10Bac感受态细胞进行重组,PCR方法验证后将重组成功的杆状病毒基因组转染sf9细胞,获得包装成功的杆状病毒,该病毒进一步接种sf9细胞,显微镜观察重组病毒引起的细胞病变,RT-PCR方法验证PEDV S1基因的mRNA表达水平,SDS-PAGE、Western blotting方法验证重组PEDV S1蛋白的表达及其反应原性。结果显示,试验成功构建了重组穿梭质粒pFastBac HT A-PEDV-S1(pSL598),成功包装表达了PEDV S1的重组杆状病毒,重组杆状病毒能使sf9细胞出现细胞变大、胞内有空泡等典型病变,PEDV S1基因的mRNA获得表达,重组蛋白His-PEDV-S1在sf9细胞中得到表达,蛋白质大小为83 ku左右,主要存在于细胞沉淀中,表达的重组蛋白能与小鼠抗His抗体和猪抗PEDV阳性血清反应,说明该蛋白具有较好的反应原性。本研究为研制PEDV新流行毒株新型亚单位疫苗和防控该毒株的流行提供了材料。
In order to study the eukaryotic expression and reactivity of porcine epidemic diarrhea virus(PEDV) isolated strain(PEDV/LA/2014/02) S1 gene,the recombinant protein(His-PEDV-S1) was expressed by the baculovirus expression system.The rare codon of PEDV S1 gene in sf9 cells was analyzed using online software,and the optimized gene was synthetized by biotechnology company.PEDV S1 gene were cloned into the baculovirus shuttle vector(pFastBac HT A),the PEDV S1 gene was recombined into baculovirus genome in Esherichia coli DH10 Bac cells and identied by PCR.The PEDV-S1 recombinant successfully baculovirus genome was transfected into sf9 cells.The packaging viruses were inoculated into sf9 cells,the cytopathic effect was observed by optical microscope.The mRNA level of PEDV S1 gene in sf9 cells was analyzed by RT-PCR.The expression and antigenicity of recombinant PEDV S1 were verificated by SDS-PAGE and Western blotting.The results showed that the pFastBac HT A-PEDV-S1(pSL598) was successfully constructed,the recombinant baculovirus expressing His-PEDV-S1 was packaged,the sf9 cells became big and round,the vacuoles appeared in cytoplasm after baculovirus infection.The mRNA of PEDV S1 gene was expressed in baculovirus infected cells.The recombinant protein His-PEDV-S1 was mainly expressed in sf9 cells precipitation,and the size of the protein was about 83 ku.The His-PEDV-S1 had good reactivity of mouse anti-His antibody and swine anti-PEDV positive antisera.This study provided materials for the development of a new subunit vaccine for the new PEDV strain and the prevention and control of PEDV strain.
In order to study the eukaryotic expression and reactivity of porcine epidemic diarrhea virus(PEDV) isolated strain(PEDV/LA/2014/02) S1 gene,the recombinant protein(His-PEDV-S1) was expressed by the baculovirus expression system.The rare codon of PEDV S1 gene in sf9 cells was analyzed using online software,and the optimized gene was synthetized by biotechnology company.PEDV S1 gene were cloned into the baculovirus shuttle vector(pFastBac HT A),the PEDV S1 gene was recombined into baculovirus genome in Esherichia coli DH10 Bac cells and identied by PCR.The PEDV-S1 recombinant successfully baculovirus genome was transfected into sf9 cells.The packaging viruses were inoculated into sf9 cells,the cytopathic effect was observed by optical microscope.The mRNA level of PEDV S1 gene in sf9 cells was analyzed by RT-PCR.The expression and antigenicity of recombinant PEDV S1 were verificated by SDS-PAGE and Western blotting.The results showed that the pFastBac HT A-PEDV-S1(pSL598) was successfully constructed,the recombinant baculovirus expressing His-PEDV-S1 was packaged,the sf9 cells became big and round,the vacuoles appeared in cytoplasm after baculovirus infection.The mRNA of PEDV S1 gene was expressed in baculovirus infected cells.The recombinant protein His-PEDV-S1 was mainly expressed in sf9 cells precipitation,and the size of the protein was about 83 ku.The His-PEDV-S1 had good reactivity of mouse anti-His antibody and swine anti-PEDV positive antisera.This study provided materials for the development of a new subunit vaccine for the new PEDV strain and the prevention and control of PEDV strain.
引文
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[8] LIU C,TANG J,MA Y,et al.Receptor usage and cell entry of porcine epidemic diarrhea coronavirus[J].Journal of Virology,2015,89(11):6121-6125.
[9] DENG F,YE G,LIU Q,et al.Identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains[J].Viruses,2016,8(3):55.
[10] LI W,VAN KUPPEVELD F J,HE Q,et al.Cellular entry of the porcine epidemic diarrhea virus[J].Virus Research,2016,226:117-127.
[11] GERDTS V,ZAKHARTCHOUK A.Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses[J].Veterinary Microbiology,2017,206:45-51.
[12] ZHAO P,WANG B,JI C M,et al.Identification of a peptide derived from the heptad repeat 2 region of the porcine epidemic diarrhea virus (PEDV) spike glycoprotein that is capable of suppressing PEDV entry and inducing neutralizing antibodies[J].Antiviral Research,2018,150:1-8.
[13] LI Q,XU Z,WU T,et al.A flagellin-adjuvanted PED subunit vaccine improved protective efficiency against PEDV variant challenge in pigs[J].Vaccine,2018,36(29):4228-4235.
[14] TUN H M,CAI Z,KHAFIPOUR E.Monitoring survivability and infectivity of porcine epidemic diarrhea virus (PEDV) in the infected on-farm earthen manure storages (EMS)[J].Front Microbiology,2016,7:265.
[15] BEALL A,YOUNT B,LIN C M,et al.Characterization of a pathogenic full-length cDNA clone and transmission model for porcine epidemic diarrhea virus strain PC22A[J].MBio,2016,7(1):e01451-15.
[16] WANG X N,WANG L,ZHENG D Z,et al.Oral immunization with a Lactobacillus casei-based anti-porcine epidemic diarrhoea virus (PEDV) vaccine expressing microfold cell-targeting peptide Co1 fused with the COE antigen of PEDV[J].Journal of Appllied Microbiology,2018,124(2):368-378.
[17] KIM S H,CHO B H,LEE K Y,et al.N-terminal domain of the spike protein of porcine epidemic diarrhea virus as a new candidate molecule for a mucosal vaccine[J].Immune Network,2018,18(3):e21.
[18] ZHAO P D,TAN C,DONG Y,et al.Genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern China from 2011 to 2013[J].Canadian Journal Veterinary Research,2015,79(1):8-15.
[19] WANG X Y,JI C J,ZHANG X,et al.Infection,genetic and virulence characteristics of porcine epidemic diarrhea virus in northwest China[J].Infection Genetics and Evolution,2018,11(1):34-39.
[20] PREMANAND B,ZHONG WEE P,PRABAKARAN M.Baculovirus surface display of immunogenic proteins for vaccine development[J].Viruses,2018,10(6):E298.
[21] WANG W,CHEN X,XUE C,et al.Production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus GP5 protein[J].Vaccine,2012,30(49):7072-7077.
[22] ROLDAO A,MELLADO M C,CASTILHO L R,et al.Virus-like particles in vaccine development[J].Expert Review Vaccines,2010,9(10):1149-1176.
[23] CHI J N,WU C Y,CHIEN M S,et al.The preparation of porcine circovirus type 2 (PCV2) virus-like particles using a recombinant pseudorabies virus and its application to vaccine development[J].Journal of Biotechnology,2014,181(3):12-19.
[24] GE J,AN Q,SONG S,et al.Construction of recombinant baculoviruses expressing infectious bursal disease virus main protective antigen and their immune effects on chickens[J].PLoS One,2015,10(7):e0132993.
[25] QI J,LIU T,LI Z,et al.Expression of limulus factor C in silkworm larvae by Bac-to-Bac/BmNPV baculovirus expression system[J].Sheng Wu Gong Cheng Xue Bao,2014,30(10):1594-1601.
[26] LIU Z,LIU Y,ZHANG Y,et al.Surface displaying of swine IgG1 Fc enhances baculovirus-vectored vaccine efficacy by facilitating viral complement escape and mammalian cell transduction[J].Veterinary Research,2017,48(1):29.
[27] HE Y,LI J,HECK S,et al.Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein:Implication for vaccine design[J].Jounal of Virology,2006,80(12):5757-5767.
[28] LI C,LI W,LUCIO DE ESESARTE E,et al.Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies[J].Jounal of Virology,2017,91(12):e00273-17.
[29] OKDA F A,LAWSON S,SINGREY A,et al.The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes[J].Virology,2017,509:185-194.
[2] CHUNG H C,NGUYEN V G,MOON H J,et al.Isolation of porcine epidemic diarrhea virus during outbreaks in south Korea,2013-2014[J].Emerging Infectious Diseases,2015,21(12):2238-2240.
[3] ZHAO X,LI Z,ZENG X,et al.Sequence analysis of the spike gene of porcine epidemic diarrhea virus isolated from South China during 2011-2015[J].Journal of Veterinary Science,2017,18(2):237-243.
[4] LANGEL S N,PAIM F C,LAGER K M,et al.Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV):Historical and current concepts[J].Virus Research,2016,226:93-107.
[5] SONG D,CHEN Y,PENG Q,et al.Full-length genome sequence of a variant porcine epidemic diarrhea virus strain,CH/GDZQ/2014,responsible for a severe outbreak of diarrhea in piglets in Guangdong,China,2014[J].Genome Announcements,2014,2(6):e01239-14.
[6] DIEP N V,NORIMINE J,SUEYOSHI M,et al.Novel porcine epidemic diarrhea virus (PEDV) variants with large deletions in the spike (S) gene coexist with PEDV strains possessing an intact S gene in domestic pigs in Japan:A new disease situation[J].PLoS One,2017,12(1):e0170126.
[7] DE HAAN C A,HAIJEMA B J,SCHELLEN P,et al.Cleavage of group 1 coronavirus spike proteins:How furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation[J].Journal of Virology,2008,82(12):6078-6083.
[8] LIU C,TANG J,MA Y,et al.Receptor usage and cell entry of porcine epidemic diarrhea coronavirus[J].Journal of Virology,2015,89(11):6121-6125.
[9] DENG F,YE G,LIU Q,et al.Identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains[J].Viruses,2016,8(3):55.
[10] LI W,VAN KUPPEVELD F J,HE Q,et al.Cellular entry of the porcine epidemic diarrhea virus[J].Virus Research,2016,226:117-127.
[11] GERDTS V,ZAKHARTCHOUK A.Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses[J].Veterinary Microbiology,2017,206:45-51.
[12] ZHAO P,WANG B,JI C M,et al.Identification of a peptide derived from the heptad repeat 2 region of the porcine epidemic diarrhea virus (PEDV) spike glycoprotein that is capable of suppressing PEDV entry and inducing neutralizing antibodies[J].Antiviral Research,2018,150:1-8.
[13] LI Q,XU Z,WU T,et al.A flagellin-adjuvanted PED subunit vaccine improved protective efficiency against PEDV variant challenge in pigs[J].Vaccine,2018,36(29):4228-4235.
[14] TUN H M,CAI Z,KHAFIPOUR E.Monitoring survivability and infectivity of porcine epidemic diarrhea virus (PEDV) in the infected on-farm earthen manure storages (EMS)[J].Front Microbiology,2016,7:265.
[15] BEALL A,YOUNT B,LIN C M,et al.Characterization of a pathogenic full-length cDNA clone and transmission model for porcine epidemic diarrhea virus strain PC22A[J].MBio,2016,7(1):e01451-15.
[16] WANG X N,WANG L,ZHENG D Z,et al.Oral immunization with a Lactobacillus casei-based anti-porcine epidemic diarrhoea virus (PEDV) vaccine expressing microfold cell-targeting peptide Co1 fused with the COE antigen of PEDV[J].Journal of Appllied Microbiology,2018,124(2):368-378.
[17] KIM S H,CHO B H,LEE K Y,et al.N-terminal domain of the spike protein of porcine epidemic diarrhea virus as a new candidate molecule for a mucosal vaccine[J].Immune Network,2018,18(3):e21.
[18] ZHAO P D,TAN C,DONG Y,et al.Genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern China from 2011 to 2013[J].Canadian Journal Veterinary Research,2015,79(1):8-15.
[19] WANG X Y,JI C J,ZHANG X,et al.Infection,genetic and virulence characteristics of porcine epidemic diarrhea virus in northwest China[J].Infection Genetics and Evolution,2018,11(1):34-39.
[20] PREMANAND B,ZHONG WEE P,PRABAKARAN M.Baculovirus surface display of immunogenic proteins for vaccine development[J].Viruses,2018,10(6):E298.
[21] WANG W,CHEN X,XUE C,et al.Production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus GP5 protein[J].Vaccine,2012,30(49):7072-7077.
[22] ROLDAO A,MELLADO M C,CASTILHO L R,et al.Virus-like particles in vaccine development[J].Expert Review Vaccines,2010,9(10):1149-1176.
[23] CHI J N,WU C Y,CHIEN M S,et al.The preparation of porcine circovirus type 2 (PCV2) virus-like particles using a recombinant pseudorabies virus and its application to vaccine development[J].Journal of Biotechnology,2014,181(3):12-19.
[24] GE J,AN Q,SONG S,et al.Construction of recombinant baculoviruses expressing infectious bursal disease virus main protective antigen and their immune effects on chickens[J].PLoS One,2015,10(7):e0132993.
[25] QI J,LIU T,LI Z,et al.Expression of limulus factor C in silkworm larvae by Bac-to-Bac/BmNPV baculovirus expression system[J].Sheng Wu Gong Cheng Xue Bao,2014,30(10):1594-1601.
[26] LIU Z,LIU Y,ZHANG Y,et al.Surface displaying of swine IgG1 Fc enhances baculovirus-vectored vaccine efficacy by facilitating viral complement escape and mammalian cell transduction[J].Veterinary Research,2017,48(1):29.
[27] HE Y,LI J,HECK S,et al.Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein:Implication for vaccine design[J].Jounal of Virology,2006,80(12):5757-5767.
[28] LI C,LI W,LUCIO DE ESESARTE E,et al.Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies[J].Jounal of Virology,2017,91(12):e00273-17.
[29] OKDA F A,LAWSON S,SINGREY A,et al.The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes[J].Virology,2017,509:185-194.