摘要
猪细小病毒(PPV)VP2蛋白N端连续9个甘氨酸富集的编码区是VP3蛋白的切割位点,常规PCR扩增容易导致该区段的缺失,为研究该缺失对PPV病毒样颗粒(VLPs)的影响,探索VP2病毒样颗粒上适合外源基因插入的位点,构建了该区段缺失的VP2的真核表达载体pCI-△VP2,并以完整VP2作为对照,采用脂质体介导法转染Vero细胞,通过生物信息学技术、SDS-PAGE、Western blotting、间接免疫荧光以及正染和免疫电镜对表达产物进行分析观察;进一步将重组质粒以核酸疫苗的方式直接肌注免疫小鼠,采用间接ELISA试验、淋巴细胞增殖试验和T细胞亚群流式细胞技术,分析免疫小鼠的体液和细胞免疫应答。结果显示,缺失△VP2和完整VP2在Vero细胞中均能自我装配成VLPs,并具有与完整病毒粒子类似血凝性,pCI-△VP2和pCI-VP2均可诱导小鼠产生较强的特异性体液免疫应答和良好的细胞免疫应答。结果表明,甘氨酸富集区的缺失不影响VP2病毒样颗粒的装配和免疫原性,△VP2同样可进行PPV VLPs疫苗和抗原转运载体的研制,为VLPs载体改造和修饰位点的探索提供了新方向,为VP2基因结构与蛋白质功能的关系提供了新的理论依据。
The N-terminal of porcine parvovirus （PPV） viral protein 2 （VP2） links a glycine-rich domain which is a cleavage site of PPV VP3.In order to conf＇n-m that the glycine-rich domain was essential for the self-assembling of virus-like particles （VLPs）.The VP2 gene with glycine-rich domain deleted and the complete VP2 gene were inserted to eukaryotie expression vector pCI-neo andwere named pCI-~VP2 and pCI-VP2. Then, pCI-AVP2, pCI-VP2 and pCI-neo were transferred into Wero Cells by liposome and the VLPs was detected by SDS-PAGE, Western blotting, indirect immunofluorescence and immunoelectron microscopy. Furthermore, 56 female Kunming mice were divided into 5 groups and injected intramuscularly with pCI-AVP2, pCI-VP2 and pCI-neo as DNA vaccine, PPV inactivated vaccine and normal saline separately. The peripheral blood of the mice was collected to analyze the subgroups of the peripheral blood mononuclear cell by flow cytometry, to detect the antibody and lymphocyte proliferation by indirect-ELISA and MTT assay separately. The results show that the VLPs were observed both in the pCI-AVP2 and pCI-VP2 transferred Vero Cells. The two VLPs could agglutinate guinea pig erythrocytes. The results also show that both the pCI-AVP2 and pCI-VP2 vaccine induced special cellular and humoral immunity effectively. Those results revealed that the glycine-rich domain is not essential for the VPL＇s self-assembling. This study provides a new theoretical evidence for the relationship between the gene structure and protein function of VP2.
出处
《生物工程学报》
CAS
CSCD
北大核心
2011年第12期1729-1741,共13页
Chinese Journal of Biotechnology
基金
教育部长江学者和创新团队发展计划(No.IRT0555)资助~~
关键词
猪细小病毒
VP2
甘氨酸富集区
缺失
病毒样颗粒
porcine parvovirus （PPV）, VP2, glycine-rich region, deletion, virus-like particles （VLPs）
