Loop EF区嵌合猪细小病毒B细胞表位对猪圆环病毒2型病毒样颗粒组装的影响

Effect of chimeric porcine parvovirus B cell epitope in Loop EF region on assembly of porcine circovirus type 2 virus-like particles

Loop EF是猪圆环病毒2型(PCV2)Cap蛋白的重要Loop结构,然而它在PCV2组装过程中的作用仍不清楚。本研究通过模拟PCV2病毒样颗粒(VLPs)3D结构,比较分析PCV2和PCV1、PCV3的氨基酸序列以及预测Loop EF的突变位点,筛选出PCV2 Loop EF中可供外源表位替换或插入的capsid表面位点,即第133位丙氨酸。探索性地针对该位点设计3个突变体,分别为猪细小病毒1型(PPV1)B细胞线性表位(228QQITDA233)插入PCV2 Cap第132和133位氨基酸、第133和134位氨基酸之间及替换第133位氨基酸。利用over-lap PCR等技术构建上述3个突变体的重组质粒,在大肠杆菌(BL21/DE3)中表达,并利用镍柱亲和层析纯化3个突变体蛋白,最后在体外进行VLPs组装,电镜结果显示3个突变体蛋白均可体外组装成完整的VLPs。结果表明,Loop EF中第133位丙氨酸的替换或插入外源表位突变不影响VLPs组装,提示PCV2 VLPs Loop EF展示外源抗原表位具有可行性。

Loop EF is an important loop region of porcine circovirus 2(PCV2) capsid protein(Cap).However it is still unclear what role Loop EF plays in the PCV2 Cap assembly process.We screened the site(133th Alanine) that could be used for substitution or insertion of exogenous epitopes by analyzing the 3D mimic structure of PCV2 virus-like particles(VLPs),comparing and analyzing the amino acid sequences of PCV1,PCV2 and PCV3,and predicting the mutation site of PCV2 Loop EF region.Three mutants based on the site(133th Alanine) of Loop EF were designed,the linear epitopes of porcine parvovirus(PPV1) B cells(228QQITDA233) was inserted between 132 th and 133 th amino acid,between 133 th and 134 th amino acid,respectively,and the 133 th amino acid was replaced by this PPV epitope.These recombinant plasmids were constructed by over-lap PCR,then expressed in E.coli(BL21/DE3),respectively.The proteins of mutants were purified by Nickel column affinity chromatography,respectively.The assembly of VLPs in vitro and the results of electron microscopy showed that the three mutant proteins could self-assemble into complete VLPs.These results confirmed that the mutation of the 133 th amino acid in Loop EF did not affect the assembly of VLPs,and it is feasible that loop EF is used to display foreign antigen epitopes.