猪瘟病毒E2蛋白配体表位的筛选与鉴定
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摘要
为进一步精确定位猪瘟病毒(CSFV)E2蛋白配体表位信息,利用DNASIS(Ver.2.5)软件分析设计合成7条多肽,主要覆盖E2蛋白,分别命名为SE21、SE22、SE231、SE232、SE241、SE242、SE03,同时设计合成1条无关多肽CP,均标记FITC。将PK-15细胞作为靶细胞,通过多肽与PK-15细胞结合试验、多肽抑制CSFV感染PK-15细胞试验和CSFV阻断多肽与PK-15细胞结合试验,筛选和鉴定E2蛋白配体表位。结果表明,多肽SE241、SE242与PK-15细胞结合的平均荧光强度分别为74.43±4.59和83.60±3.11,显著高于其他多肽,抑制CSFV感染PK-15细胞的感染抑制率也显著高于其他多肽,同时这2条多肽与PK-15细胞的结合被CSFV有效阻断。证实SE241、SE242多肽为CSFV与靶细胞PK-15细胞结合的配体表位,并能抑制CSFV感染PK-15细胞。本试验为CSFV新型多肽疫苗或免疫佐剂的研制提供依据。
In order to further accurately ascertain the E2 protein ligand epitope information,seven polypeptides were designed and synthesized by DNASIS(Ver.2.5) software.They mainly covered with E2 protein of classical swine virus(CSFV),named SE21,SE22,SE231,SE232,SE241,SE242,SE03.At the same time,an unrelated polypeptide CP was designed and synthesized,and the all were labeled FITC,PK-15 cells were used as target cells.And the E2 protein ligand sites were screened and identified through the PK-15 cell binding test,inhibition of CSFV infection by peptides and binding assay of CSFV blocking peptide with PK-15 cells.Results indicated that the average fluorescence intensity of the peptide SE241 and SE242 combined with PK-15 cells were 74.43±4.59,and 83.60±3.11,which were significantly higher than that of other polypeptides.The inhibitory rate of CSFV infection on PK-15 cells was also significantly higher than that of other polypeptides and the binding of these two polypeptides to PK-15 cells was effectively blocked by CSFV.It was confirmed that SE241 and SE242 polypeptides were ligand epitopes of CSFV binding to target cells PK-15 cells,and could inhibit CSFV infection of PK-15 cells.This study further reveals the molecular basis of CSFV infection in target cells and provides theoretical support for the development of novel peptide vaccines or immune adjuvants for CSFV.
In order to further accurately ascertain the E2 protein ligand epitope information,seven polypeptides were designed and synthesized by DNASIS(Ver.2.5) software.They mainly covered with E2 protein of classical swine virus(CSFV),named SE21,SE22,SE231,SE232,SE241,SE242,SE03.At the same time,an unrelated polypeptide CP was designed and synthesized,and the all were labeled FITC,PK-15 cells were used as target cells.And the E2 protein ligand sites were screened and identified through the PK-15 cell binding test,inhibition of CSFV infection by peptides and binding assay of CSFV blocking peptide with PK-15 cells.Results indicated that the average fluorescence intensity of the peptide SE241 and SE242 combined with PK-15 cells were 74.43±4.59,and 83.60±3.11,which were significantly higher than that of other polypeptides.The inhibitory rate of CSFV infection on PK-15 cells was also significantly higher than that of other polypeptides and the binding of these two polypeptides to PK-15 cells was effectively blocked by CSFV.It was confirmed that SE241 and SE242 polypeptides were ligand epitopes of CSFV binding to target cells PK-15 cells,and could inhibit CSFV infection of PK-15 cells.This study further reveals the molecular basis of CSFV infection in target cells and provides theoretical support for the development of novel peptide vaccines or immune adjuvants for CSFV.
引文
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[14] 张秋雨,李鹏,陈晴,等.猪瘟病毒在猪体组织中的分布[J].动物医学进展,2018,39(7):6-12.
[15] LIANG D,SAINZ L F,ISRARUL H,et al.The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses[J].J Gen Virol,2003,84:1269-1274.
[2] 李栋梁,王新港,郭天准,等.2014-2015年河南省猪瘟野毒E2、E0基因变异分析[J].中国兽医学报,2017,37(7):1212-1219.
[3] CAROLINE F,OLIVER B,NICOLAS R.Classical swine fever virus replicon particles lacking the Erns gene:a potential marker vaccine for intradermal application[J].Vet Res,2006,37(5):655-670.
[4] MANFRE A J,SIMON A E.Importance of coat protein and RNA silencing in satellite RNA/virus interactions[J].Virology,2008,379(1):161-167.
[5] VELAZQUEZSALINAS L,RISATTI G R,HOLINKA L G,et al.Recoding structural glycoprotein E2 in classical swine fever virus (CSFV) produces complete virus attenuation in swine and protects infected animals against disease[J].Virology,2016,49(4):178-189.
[6] YANG Z,SHI Z,GUO H,et al.Annexin 2 is a host protein binding to classical swine fever virus E2 glycoprotein and promoting viral growth in PK-15 cells[J].Virus Res,2015,201:16-23.
[7] ASFOR A S,WAKELEY P R,DREW T W,et al.Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency[J].Virus Res,2014,189:147-157.
[8] SIEGERT S,SCHNIERLE P,SCHNIERLE B S.Novel anti-viral therapy:drugs that block HIV entry at different target sites[J].Mini Rev Med Chem,2006,6(5):557-562.
[9] CHAKRABORTY S,VEETTIL M V,CHANNDRAN B.Kaposi’s arcoma associated herpesvirus entry into target cells[J].Front Microbiol,2012(3):6.
[10] 冯春花,岳锋.猪瘟病毒受体与配体研究进展[J].中国兽医杂志,2015(9):75-78.
[11] HULST M M,MOORMANN R J.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].J Gen Virol,1997,78(11):2779-2787.
[12] WANG Z,NIE Y,WANG P,et al.Charactekization of classical swine fever virus entry by using pseudotyped viruses:E1 and E2 are sufficient to mediate viral entry[J].Virology,2004,330:332-341.
[13] MEI Y,YUE F,NING H M,et al.Identificction of the linear ligand epitope on classical swine fever virus that interacts with porcine kidney 15 cells[J].Can J Vet Res,2017,81(3):186-191.
[14] 张秋雨,李鹏,陈晴,等.猪瘟病毒在猪体组织中的分布[J].动物医学进展,2018,39(7):6-12.
[15] LIANG D,SAINZ L F,ISRARUL H,et al.The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses[J].J Gen Virol,2003,84:1269-1274.