猪瘟病毒感染靶细胞的配体表位研究
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摘要
猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起猪的高度接触性、致死性疾病。CSFV是黄病毒科、瘟病毒属成员之一,Erns、E1和E2是CSFV的三种主要结构蛋白,此三种结构蛋白与病毒的吸附和侵入靶细胞有关。为确定CSFV与病毒受体结合的具体配体表位信息,本研究利用生物信息学技术分析CSFV Erns、E1和E2三种蛋白一级结构的氨基酸序列,设计合成10条多肽,涵盖以上三种蛋白的所有序列,以PK-15细胞为靶细胞,进行多肽与靶细胞结合试验和病毒阻断试验筛选鉴定CSFV的配体表位;然后通过多肽抑制CSFV感染PK-15细胞试验测定其感染抑制功能。结果显示,E2蛋白上的一段多肽SE24在0.2mmol/L、0.1mmol/L、0.05mmol/L和0.025mmol/L浓度下能有效结合PK-15细胞;病毒阻断试验显示CSFV可以阻断多肽SE24结合PK-15细胞,与结合试验相比,阳性细胞数减少了11.6%,平均荧光强度降低了3.07;多肽抑制CSFV感染试验表明多肽SE24可以抑制CSFV感染PK-15细胞,具有感染抑制功能。通过结合试验、病毒阻断试验和感染抑制试验成功筛选出一条CSFV配体表位SE24 ,位于E2蛋白上,氨基酸序列为VHASDERLGPMPCRPKEIGSSAGPVRKTSCTFNYAKTGKNKYYEPRDSYF;并且配体表位SE24多肽能够抑制CSFV感染PK-15细胞,随着多肽浓度的增加感染率降低,具有剂量依赖性,浓度在0.2mmol/L时可以完全抑制病毒感染靶细胞。
Classical swine fever (CSF) is a highly contagious and fatal disease of swine, which is caused by Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae. Erns, E1 and E2 are the chief structrural proteins of CSFV and relevant to adsorption and invasion of target cell. For identification of the ligand epitope binding with target cells of CSFV, we designed and synthesized ten peptides according to the amino acid sequence of the glycoprotein Erns E1 and E2 of the CSFV by using bioinformatics technique. Porcine kidney 15 (PK-15) cells were used as the target cell. Peptide binding with PK-15 cell and blocked binding assays were used for screening and identification of ligand epitope of CSFV. Furthermor, CSFV infection inhibiting experiment was applyed to identify the function of the epitope peptide. The results indicate that the polypeptide SE24 in E2 glycoprotein of CSFV can bind with PK-15 cells effectively at the concentration of 0.2mmol/L, 0.1mmol/L, 0.05mmol/L and 0.025mmol/L. Blocking assay showes that the epitope peptide SE24 can inhibite the CSFV infecting PK15 cells, in which the precentage of positive cells and the mean fluorescence intensity reduced by 11.6% and 3.07, respectively. These results demonstrate that peptide SE24 is a ligand epitope of the CSFV, and its amino acid sequence is VHASDERLGPMPCRPKEIGSSAGPVRKTSCTFNYAKTGKNKYYEPRDSYF on the glycoprotein E2. It is suggested that the ligand epitope peptide SE24 has the function of infection inhibiting with dose dependent according to the infection rate of CSFV decreased while the concentration of the ploypeptide SE24 increased. And the infection of CSFV is inhibited completely at the concentration of 0.2mmol/L of the ploypeptide SE24.
Classical swine fever (CSF) is a highly contagious and fatal disease of swine, which is caused by Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae. Erns, E1 and E2 are the chief structrural proteins of CSFV and relevant to adsorption and invasion of target cell. For identification of the ligand epitope binding with target cells of CSFV, we designed and synthesized ten peptides according to the amino acid sequence of the glycoprotein Erns E1 and E2 of the CSFV by using bioinformatics technique. Porcine kidney 15 (PK-15) cells were used as the target cell. Peptide binding with PK-15 cell and blocked binding assays were used for screening and identification of ligand epitope of CSFV. Furthermor, CSFV infection inhibiting experiment was applyed to identify the function of the epitope peptide. The results indicate that the polypeptide SE24 in E2 glycoprotein of CSFV can bind with PK-15 cells effectively at the concentration of 0.2mmol/L, 0.1mmol/L, 0.05mmol/L and 0.025mmol/L. Blocking assay showes that the epitope peptide SE24 can inhibite the CSFV infecting PK15 cells, in which the precentage of positive cells and the mean fluorescence intensity reduced by 11.6% and 3.07, respectively. These results demonstrate that peptide SE24 is a ligand epitope of the CSFV, and its amino acid sequence is VHASDERLGPMPCRPKEIGSSAGPVRKTSCTFNYAKTGKNKYYEPRDSYF on the glycoprotein E2. It is suggested that the ligand epitope peptide SE24 has the function of infection inhibiting with dose dependent according to the infection rate of CSFV decreased while the concentration of the ploypeptide SE24 increased. And the infection of CSFV is inhibited completely at the concentration of 0.2mmol/L of the ploypeptide SE24.
引文
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[16] Hulst MM, Moormann RJ. Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptors [J]. Journal of general virology, 1997, 78(11): 2779-2787.
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[18] Hulst MM, Van Gennip HG, Vlot AC, et al. Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence [J]. Journal of general virology, 2001, 75 (20): 9585-9595.
[19] Hulst MM, Himes G, Newbigin E, et al. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease [J]. Virology, 1994, 200(2): 558-565.
[20] Iqbal M, John W. Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site directed mutagenesis [J]. Journal of General Virology, 2002, 83: 2153-2159.
[21] Chen L, Xia YH, Pan ZSH, et al. Expression and functional characterization of classical swine fever virus Erns protein [J]. Protein Expression and Purification, 2007, 55: 379-387.
[22] Hulst MM, Panoto FE, Hoeklnan A. Inactivation of the RNase activity of glycoprotein Erns of elassieal swine fever virus results in a cytopatllogenie virus [J]. Journal of general virology, 1998, 72(l): 151-157.
[23] Fernandez Sainz I, Holinka LG, Lu Z, et al. Removal of N-linked glycosylation site of classical swine fever virus strain Brescia Erns glycoprotein affects virulence in swine [J]. Virology, 2008, 370: 122-129.
[24] Risatti GR, Holinka LG, Lu Z. Mutation of E1 glycoprotein of classical swine fever virus affect viral virulence in swine [J]. Virology, 2005, 343: 116-127.
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