鸡传染性支气管炎病毒S1蛋白MHCⅠ分子限制性T细胞表位的鉴定
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摘要
利用生物结构技术预测传染性支气管炎S1蛋白的1条细胞毒性T淋巴细胞(CTL)表位,并合成相应的候选多肽(Sp6)。为了确定S1蛋白中确实存在该T细胞表位,并鉴定这条T细胞表位的正确性,构建了含有S1基因的重组质粒pCAGGS-S1,通过间接免疫荧光和Western blot确定该重组质粒可以在真核细胞表达后,将该重组质粒免疫SPF鸡,间接ELISA检测血清中的抗体。采集免疫鸡的脾淋巴细胞并用合成的候选多肽刺激,通过实时荧光定量PCR检测脾淋巴细胞中IFN-γ的分泌量以及流式细胞术检测CD8+T淋巴细胞的增殖情况,初步确定该候选肽的正确性。间接免疫荧光、Western blot和间接ELISA试验结果表明,S1蛋白能够在293T细胞中获得表达并能够引起机体的体液免疫,说明S1蛋白具有反应原性,为进一步验证T细胞表位打下了基础;荧光定量PCR结果显示Sp6刺激后的鸡的脾淋巴细胞中IFN-γ的分泌量明显增加;流式细胞检测发现经Sp6和不相关多肽NP89-97刺激后及空白对照,CD8+T淋巴细胞增殖分别为34.8%、2.6%、0。以上结果表明,多肽Sp6能在体外诱导活化的鸡淋巴细胞产生细胞毒性T淋巴细胞反应,是IBV S1的CTL表位。该研究结果对传染性支气管炎病毒免疫机制和通用疫苗研究具有借鉴意义。此次研究结果表明多肽Sp6能在体外诱导活化的鸡淋巴细胞产生CTL,是IBV S1的CTL表位。该研究结果对传染性支气管炎病毒免疫机制和通用疫苗研究具有借鉴意义。
A T cell epitope candidate peptide Sp6 of the avian infectious bronchitis virus S1 was predicted with biological structures technology.To identify this peptide exist in S1 protein and verify validity of Sp6,the recombinant plasmid pCAGGS-S1 was constructed.While it can be expressed in eukaryotic cells after determined by indirect immunofluorescence and Western blot,using the recombinant plasmid immune SPF chickens,and the antibodies against S1 were detectedby ELISA.Splenic cells were collected from inoculated chickens and stimulated with the peptides Sp6,NP89-97(PKKTGGPIY)and negative control.By SBRY Green qPCR detecting IFN-γproduction of spleen lymphocytes and flow cytometry detecting the multiplication of CD8+T lymphocytes,preliminarily make sure that the candidate peptide is correct.The IFA/Western blot and ELISA results showed that the expression of S1 was confirmed in 293 Tand can induced the humoral immune,indicate that S1 protein is reaction to the original and lied a foundation for further validation of T cell epitope;the results of SBRY Green qPCR showed that spleen lymphocytes of chickens stimulated with the peptide Sp6 secreted significantly-increased IFN-γ.Flow cytometry assay results showed that the CD8+T lymphocytes were proliferated by 34.8%,2.6%,and 0,respectively,which indicated that the peptide Sp6 was a T cell epitope of IBV S1 and it can induce activation of chicken lymphocytes to produce cytotoxic T lymphocyte(CTL)reaction in vivo.All above results indicate that the peptide Sp6 was a T cell epitope of IBV S1 and it can induce activation of chicken lymphocytes to produce CTL reaction in vivo.The results possesses significance to mechanism of protective immunity against avian infectious bronchitis virus and the research towards universal IBV vaccine.
A T cell epitope candidate peptide Sp6 of the avian infectious bronchitis virus S1 was predicted with biological structures technology.To identify this peptide exist in S1 protein and verify validity of Sp6,the recombinant plasmid pCAGGS-S1 was constructed.While it can be expressed in eukaryotic cells after determined by indirect immunofluorescence and Western blot,using the recombinant plasmid immune SPF chickens,and the antibodies against S1 were detectedby ELISA.Splenic cells were collected from inoculated chickens and stimulated with the peptides Sp6,NP89-97(PKKTGGPIY)and negative control.By SBRY Green qPCR detecting IFN-γproduction of spleen lymphocytes and flow cytometry detecting the multiplication of CD8+T lymphocytes,preliminarily make sure that the candidate peptide is correct.The IFA/Western blot and ELISA results showed that the expression of S1 was confirmed in 293 Tand can induced the humoral immune,indicate that S1 protein is reaction to the original and lied a foundation for further validation of T cell epitope;the results of SBRY Green qPCR showed that spleen lymphocytes of chickens stimulated with the peptide Sp6 secreted significantly-increased IFN-γ.Flow cytometry assay results showed that the CD8+T lymphocytes were proliferated by 34.8%,2.6%,and 0,respectively,which indicated that the peptide Sp6 was a T cell epitope of IBV S1 and it can induce activation of chicken lymphocytes to produce cytotoxic T lymphocyte(CTL)reaction in vivo.All above results indicate that the peptide Sp6 was a T cell epitope of IBV S1 and it can induce activation of chicken lymphocytes to produce CTL reaction in vivo.The results possesses significance to mechanism of protective immunity against avian infectious bronchitis virus and the research towards universal IBV vaccine.
引文
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[11]KUO S M,KAO H W,HOU M H,et al.Evolution of infectious bronchitis virus in Taiwan:Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity[J].Vet Microbiol,2013,162(2-4):408-418.
[12]IGNJATOVIC J,SAPATS S.Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus[J].Arch Virol,2005,150(9):1813-1831.
[13]CORR M,SLANETZ A E,BOYD L F,et al.T cell receptor-MHC class I peptide interactions:affinity,kinetics,and specificity[J].Science,1994,265(5174):946-949.
[14]BUDHIA S,HARING L F,McCONNELL I,et al.Quantitation of ovine cytokine mRNA by real-time RT-PCR[J].J Immunol Methods,2006,309(1-2):160-172.
[15]SINGH R,RECINOS R F,AGRESTI M,et al.Realtime reverse transcriptase polymerase chain reaction:an improvement in detecting mRNA levels in mouse cranial tissue[J].Plast Reconstr Surg,2006,117(7):2227-2234.
[16]SMITH R A,LEA R A,WEINSTEIN S R,et al.Detection of mRNA levels for the estrogen alpha,estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue[J].Cancer Lett,2006,237(2):248-255.
[2]LIU S W,KONG X G.A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in China[J].Avian Pathol,2004,33(3):321-327.
[3]BONNEFOY L,BOUQUET J F,PICAULT J P,et al.Characterization of IBV variant strain PL 84084isolated in France[J].Adv Exp Med Biol,1993,342:395-397.
[4]CASAIS R,DOVE B,CAVANAGH D,et al.Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism[J].J Virol,2003,77(16):9084-9089.
[5]LAMBERT L C,FAUCI A S.Influenza vaccines for the future[J].N Engl J Med,2010,363(21):2036-2044.
[6]ZHOU J Y,CHENG L Q,ZHENG X J,et al.Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus[J].J Biotechnol,2004,111(2):121-130.
[7]CHEN B Y,ITAKURA C.Cytopathology of chick renal epithelial cells experimentally infected with avian infectious bronchitis virus[J].Avian Pathol,1996,25(4):675-690.
[8]田占成,孙永科,王云峰,等.表达鸡传染性支气管炎病毒S1基因重组鸡痘病毒对SPF鸡的免疫保护作用[J].畜牧兽医学报,2006,37(6):580-586.TIAN Z C,SUN Y K,WANG Y F,et al.The immunological efficacies of recombinant fowlpox virus expressing the S1gene of LX4strain of infections bronchitis virus in specific pathogen free(SPF)chickens[J].Acta Veterinaria et Zootechnica Sinica,2006,37(6):580-586.(in Chinese)
[9]CORR M,SLANETZ A E,BOYD L F,et al.T cell receptor-MHC class I peptide interactions:affinity,kinetics,and specificity[J].Sciences,1994,265(5174):946-949.
[10]侯艳霞,郭莹莹,沈楠,等.一株H5亚型禽流感病毒NP蛋白两个鸡MHC分子限制性T细胞表位的鉴定[J].中国预防兽医学报,2011,33(6):457-460.HOU Y X,GUO Y Y,SHEN N,et al.Identification of two T cell epitopes of nucleoprotein from an H5avian influenza virus in chickens[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(6):457-460.(in Chinese)
[11]KUO S M,KAO H W,HOU M H,et al.Evolution of infectious bronchitis virus in Taiwan:Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity[J].Vet Microbiol,2013,162(2-4):408-418.
[12]IGNJATOVIC J,SAPATS S.Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus[J].Arch Virol,2005,150(9):1813-1831.
[13]CORR M,SLANETZ A E,BOYD L F,et al.T cell receptor-MHC class I peptide interactions:affinity,kinetics,and specificity[J].Science,1994,265(5174):946-949.
[14]BUDHIA S,HARING L F,McCONNELL I,et al.Quantitation of ovine cytokine mRNA by real-time RT-PCR[J].J Immunol Methods,2006,309(1-2):160-172.
[15]SINGH R,RECINOS R F,AGRESTI M,et al.Realtime reverse transcriptase polymerase chain reaction:an improvement in detecting mRNA levels in mouse cranial tissue[J].Plast Reconstr Surg,2006,117(7):2227-2234.
[16]SMITH R A,LEA R A,WEINSTEIN S R,et al.Detection of mRNA levels for the estrogen alpha,estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue[J].Cancer Lett,2006,237(2):248-255.