猪圆环病毒2b型病毒样颗粒表面可用于外源肽展示的位点分析  

Site analysis of Porcine circovirus type 2b virus-like particle surface for exogenous peptide display

作　　者：李国攀[1] 荣俊[1] 谢明 匡红艳[1] LI Guopan;RONG Jun;XIE Ming;KUANG Hongyan(College of Life Science,Yangtze University,Jingzhou 434020,China)

机构地区：[1]长江大学生命科学学院,湖北荆州434020

出　　处：《黑龙江畜牧兽医》2022年第17期12-18,140,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：动物基因工程疫苗国家重点实验室开放基金项目“猪圆环病毒3型感染性克隆毒株制备”(SKLGEVV-ZD/ZY-2019)。

摘　　要：为了分析猪圆环病毒2b型病毒样颗粒(virus-like particles of Porcine circovirus type 2b,PCV-2b VLPs)表面具有外源肽展示潜力的位点,试验通过PyMol软件分析了PCV-2b VLPs表面的位点分布,并以高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)Gp5的中和表位B(Epitope B,EpB)为外源肽,通过重叠PCR方法将表面位点替换为EpB,构建EpB嵌合的PCV-2b衣壳蛋白(Cap),利用原核表达系统诱导表达后进行凝胶层析纯化和Western-blot检测,并利用透射电子显微镜分析VLPs的组装状态。结果表明:在PCV-2b VLPs表面共筛选到8个可用于外源肽展示的位点,分别为S1(Y^(50)TIKRTTVKTP^(60))、S2(L^(75)PPGGGSN^(82))、S3(F^(124)VTKATAL^(131))、S4(V^(161)LDSTIDY^(168))、S5(Q^(183)TAGNVD^(189))、S6(I^(201)YDQE^(205))、S7(K^(222)DPPL^(226))、S8(S^(104)PITQGDRGVGS^(115))位点;将EpB替换展示到S1~S8位点后,构建的EpB嵌合的PCV-2b Cap能够在原核表达系统中获得不同程度的可溶性表达,其中S1~S7位点被EpB替换后能够实现较高水平的可溶性表达;8个目的蛋白均能被抗EpB抗体特异性识别,也均能够自组装形成VLPs,但粒径大小和形态与PCV-2b VLPs有不同程度的差异。说明试验筛选出的8个位点均有一定的外源肽展示能力。In order to analyze sites on the surface of virus-like particles of porcine circovirus type 2 b(PCV-2 b VLPs)which had the potential to display exogenous peptides,in the experiment,the site distribution on the surface of PCV-2 b VLPs was analyzed by PyMol software.The neutralizing epitope B(EpB)of highly pathogenic Porcine reproductive and respiratory syndrome virus Gp5 was used as exogenous peptide,and the surface site was replaced by EpB by overlapping PCR method to construct EpB-chimeric PCV-2 b Cap.After induced expression by prokaryotic expression system,gel chromatography purification and western blot analysis were performed,and the assembly status of virus-like particles was analyzed by transmission electron microscopy.The results showed that eight sites that could be used for exogenous peptide display were screened:S1(Y^(50)TIKRTTVKTP^(60)),S2(L^(75)PPGGGSN^(82)),S3(F^(124)VTKATAL^(131)),S4(V^(161)LDSTIDY^(168)),S5(Q^(183)TAGNVD^(189)),S6(I^(201)YDQE^(205)),S7(K^(222)DPPL^(226))and S8(S^(104)PITQGDRGVGS^(115)),respectively.After the EpB replacement was displayed at the S1-S8 sites,the constructed EpB chimeric PCV-2 b Cap could obtain different degrees of soluble expression in the prokaryotic expression system;the S1-S7 sites were replaced by EpB to achieve a higher level of soluble expression.The eight proteins constructed and expressed could be specifically recognized by anti-EpB antibodies and could self-assemble to form virus-like particles,but the particle size and shape were different from PCV-2 b VLPs to varying degrees.The results suggested that the 8 sites screened out in the experiment had certain exogenous peptide display ability.

关 键 词：猪圆环病毒2b型 病毒样颗粒 表面展示 原核表达系统 嵌合病毒样颗粒 

分 类 号：S852[农业科学—基础兽医学]