猪轮状病毒VP7蛋白模拟表位的噬菌体展示技术筛选与鉴定
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摘要
猪轮状病毒(Po RV)是引起仔猪腹泻的重要病原之一,其主要结构蛋白衣壳蛋白VP7可诱导机体产生中和抗体。文章以制备的抗Po RV VP7单克隆抗体(MAb)3B6作为靶分子,利用噬菌体十二肽库展示技术对其模拟表位进行筛选。四轮筛选后随机挑取阳性克隆扩增后进行ELISA鉴定,同时提取其基因组DNA测序,10个阳性噬菌体亲和肽拥有共同的外源肽序列"VPLGTDNGDIWV",而且与靶分子有很高亲和力。阳性噬菌体亲和肽与VP7蛋白进行序列比对,结果表明,十二肽中有4个氨基酸(第167位P、第178位T、第179位D和第184位W)与VP7蛋白167~184位氨基酸有一定的相似性。竞争抑制ELISA证明亲和多肽降低Po RV与抗VP7单克隆抗体之间的作用,病毒竞争抑制试验表明亲和多肽可以竞争性地抑制Po RV感染细胞。因此由这4个氨基酸构成的基序很有可能作为猪轮状病毒VP7蛋白的一个模拟表位。
Porcine rotavirus(Po RV) causes diarrhea, vomit, dehydration, acid- base imbalance and death in piglets. Po RV VP7 is the outer capsid glycoprotein that is highly immunogenic and induce the production of neutralizing antibody. A prepared monoclonal antibody(MAb) 3B6 against Po RV VP7 protein was used as immobilized target in a subtract biopanning process using a 12-mer phage display random peptide library. After four rounds of pannings, 10 phages bearing the consensus peptide VPLGTDNGDIWV and having a specific binding activity to the target were identified. Sequence alignment between positive phage-displayed 12-mer peptide and VP7 protein suggested that a potential motif that is identical with 167(P), 178(T), 179(D) or 184(W) aa in the VP7 protein. Competition ELISA showed that the high affinity peptide reduced the combination of Po RV with anti- VP7 MAb. Moreover,this peptide could significantly inhibit Po RV propagation in cell culture. In conclusion, the 4 amino acid motif(P-TD-W) functions as a spatial conformation epitope of Po RV VP7 protein.
Porcine rotavirus(Po RV) causes diarrhea, vomit, dehydration, acid- base imbalance and death in piglets. Po RV VP7 is the outer capsid glycoprotein that is highly immunogenic and induce the production of neutralizing antibody. A prepared monoclonal antibody(MAb) 3B6 against Po RV VP7 protein was used as immobilized target in a subtract biopanning process using a 12-mer phage display random peptide library. After four rounds of pannings, 10 phages bearing the consensus peptide VPLGTDNGDIWV and having a specific binding activity to the target were identified. Sequence alignment between positive phage-displayed 12-mer peptide and VP7 protein suggested that a potential motif that is identical with 167(P), 178(T), 179(D) or 184(W) aa in the VP7 protein. Competition ELISA showed that the high affinity peptide reduced the combination of Po RV with anti- VP7 MAb. Moreover,this peptide could significantly inhibit Po RV propagation in cell culture. In conclusion, the 4 amino acid motif(P-TD-W) functions as a spatial conformation epitope of Po RV VP7 protein.
引文
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[17]Malhotra U,Nolin J,Horton H,et al.Functional properties and epitope characteristics of T-cells recognizing natural HIV-1variants[J].Vaccine,2009,27(48):6678-6687.
[2]Neog B K,Barman N N,Bora D P,et al.Experimental infection of pigs with group A rotavirus and enterotoxigenic Escherichia coli in India:gross,histopathological and immunopathological study[J].Vet Ital,2011,47(2):117-128.
[3]师东方,李一经,王君伟,等.猪轮状病毒与传染性胃肠炎病毒核酸免疫研究[J].东北农业大学学报,2006,36(5):605-610.
[4]Tatte V S,Rawal K N,Chitambar S D.Sequence and phylogenetic analysis of the VP6 and NSP4 genes of human rotavirus strains:evidence of discordance in their genetic linkage[J].Infection,Genetics and Evolution,2010,10(7):940-949.
[5]Wang L F,Yu M.Epitope identification and discovery using phage display libraries:applications in vaccine development and diagnostics[J].Current Drug Targets,2004,5(1):1-15.
[6]Jiang L,Zhou J M,Yin Y,et al.Selection and identification of Bcell epitope on NS1 protein of dengue virus type 2[J].Virus Research,2010,150(1):49-55.
[7]徐和敏,王琴,徐璐,等.猪瘟病毒E2基因噬菌体展示多肽库的构建及表位鉴定[J].畜牧兽医学报,2010,41(1):71-76.
[8]Pedroza-Roldan C,Charles-Nio C,Saavedra R,et al.Variable表位的噬菌体展示技术筛选与鉴定·17·epitope library-based vaccines:Shooting moving targets[J].Molecular Immunology,2009,47(2):270-282.
[9]杨巍,李广兴,孟凡丹,等.猪流行性腹泻病毒N基因克隆及原核表达[J].东北农业大学学报,2011,42(6):81-85.
[10]洪琴,任晓峰,李广兴.鸡痘DNA疫苗Pvax-env构建及其体内表达[J].东北农业大学学报,2011,42(6):105-108.
[11]石娜,李广兴,任晓峰,等.猪IL-15与PRRS病毒GP5基因核酸疫苗免疫效力[J].东北农业大学学报,2010,41(1):97-102.
[12]Eswar N,Webb B,Marti-Renom M A,et al.Comparative protein structure modeling using Modeller[J].Current Protocols in Bioinformatics,2006:5.6.1-5.6.30.
[13]Martí-Renom M A,Stuart A C,Fiser A,et al.Comparative protein structure modeling of genes and genomes[J].Annual review of biophysics and biomolecular structure,2000,29(1):291-325.
[14]赵高伟,任晓峰.轮状病毒感染机制及防治的研究进展[J].世界华人消化杂志,2013,1:021.
[15]时洪艳,冯力,陈建飞,等.猪轮状病毒OSU强毒株VP7基因的克隆及其抗原表位原核表达和免疫学活性分析[J].中国预防兽医学报,2008,30(2):136-140.
[16]Jiang L,Zhou J M,Yin Y,et al.Selection and identification of Bcell epitope on NS1 protein of dengue virus type 2[J].Virus research,2010,150(1):49-55.
[17]Malhotra U,Nolin J,Horton H,et al.Functional properties and epitope characteristics of T-cells recognizing natural HIV-1variants[J].Vaccine,2009,27(48):6678-6687.