猪圆环病毒2型病毒样颗粒三倍轴区域嵌合猪细小病毒抗原表位的展示策略及免疫原性

doi:10.11843/j.issn.0366-6964.2025.01.028

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (1): 307-318.doi: 10.11843/j.issn.0366-6964.2025.01.028

猪圆环病毒2型病毒样颗粒三倍轴区域嵌合猪细小病毒抗原表位的展示策略及免疫原性

蔡云凤¹^,²(), 李宏¹^,², 黄小铭¹^,², 何庆¹^,², 丁振宇¹^,², 余婉婷³, 罗施乐⁴, 陈方志⁵, 王乃东¹^,², 杨毅¹^,², 湛洋¹^,²^,*()

1. 湖南农业大学动物医学院, 长沙 410128
2. 兽用蛋白质工程疫苗湖南省重点实验室, 长沙 410128
3. 江西农业大学动物科学技术学院, 南昌 330045
4. 湖南派智生物科技有限公司, 长沙 410205
5. 宁乡市动物疫病预防控制中心, 长沙 410600

收稿日期:2024-02-19 出版日期:2025-01-23 发布日期:2025-01-18
通讯作者: 湛洋 E-mail:Yvonne_2019@stu.hunau.edu.cn;yangzhan@hunau.edu.cn
作者简介:蔡云凤(1997-)，女，江西丰城人，硕士，主要从事动物病毒蛋白质组学研究, E-mail: Yvonne_2019@stu.hunau.edu.cn
基金资助:
国家自然科学基金项目(32202790)；湖南省自然科学基金项目(2022JJ40183)；湖南省技术攻关“揭榜挂帅”项目(2021NK1030)；湖南省青年科技人才项目(2022RC1046)；湖南省教育厅科学研究重点项目(23A0194)

Study on the Presentation Strategy and Immunogenicity of Porcine Parvovirus Epitope Inserted by 3-fold Axes Region on the Surface of Porcine Circovirus Type 2 Virus-like Particles

CAI Yunfeng¹^,²(), LI Hong¹^,², HUANG Xiaoming¹^,², HE Qing¹^,², DING Zhenyu¹^,², YU Wanting³, LUO Shile⁴, CHEN Fangzhi⁵, WANG Naidong¹^,², YANG Yi¹^,², ZHAN Yang¹^,²^,*()

1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China
2. Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Changsha 410128, China
3. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China
4. Hunan Panwise Biotechnology Co., LTD, Changsha 410205, China
5. Animal Disease Prevention and Control Center of Ningxiang, Changsha 410600, China

Received:2024-02-19 Online:2025-01-23 Published:2025-01-18
Contact: ZHAN Yang E-mail:Yvonne_2019@stu.hunau.edu.cn;yangzhan@hunau.edu.cn

摘要/Abstract

摘要：

为了探索猪圆环病毒2型(porcine circovirus type 2, PCV2)病毒样颗粒三倍轴区域展示外源抗原表位的潜力，本研究借助结构模拟和蛋白质的三维结构展示，分析了PCV2核衣壳表面氨基酸残基突变后的结构差异。通过分析不同cap基因突变质粒在大肠杆菌表达系统中所表达的突变体蛋白的可溶性和纯化难易程度，确定合适的外源表位嵌合区。将嵌合PPV抗原表位的Cap蛋白经体外组装获得重组的病毒样颗粒，将此病毒样颗粒免疫小鼠，通过抗体检测来评价其免疫原性。结果显示，PCV2核衣壳表面三倍轴区域存在两个适合用于展示外源表位的氨基酸区域(分别命名为Motif A和Motif B)；相同表达条件下，Motif A区域的突变蛋白均以可溶性表达为主，且能一步纯化获得目的蛋白，而Motif B区域的突变蛋白多以包涵体形式存在；Motif A区域嵌合PPV表位后，纯化的重组蛋白能在体外组装成cVLPs，并通过间接免疫荧光试验证明其保留有野生型VLPs内化PK15细胞的能力；形成的cVLPs能刺激小鼠机体产生分别针对PCV2 VLPs、B5-E1表位和PPV VP2蛋白的抗体。综上表明，PCV2 Cap蛋白Loop GH区域Motif A能够将外源表位展示在病毒样颗粒外表面，并且能够被机体免疫细胞识别产生特异性抗体。

关键词: 猪圆环病毒2型, 病毒样颗粒, 三倍轴区域, 外源表位

Abstract:

In order to study the potential of displaying foreign epitopes in the 3-fold axes region on the surface of PCV2 capsid, 3D structures of recombinant proteins after amino acid mutation are modeled, displayed and analyzed, combined with PCV2 capsid surface amino acid analysis. The constructed plasmids were expressed in E.coli (BL21), and the differences in soluble expression and purification difficulty of various mutant cap genes were analyzed, then the suitable region for displaying foreign epitope was determined. After the recombinant Cap protein inserted with PPV epitope was assembled in vitro, then the chimeric VLPs were immunized to mice, and the immunogenicity of chimeric VLPs was evaluated by antibody detection. The results showed that there were two amino acid regions (named Motif A and Motif B) suitable for displaying exogenous epitopes in 3-fold axes region on the surface of PCV2 capsid. Under the same expression conditions, mutant proteins related to Motif A region were mainly expressed in supernatant, and the target protein could be purified in one step, while most mutant proteins related to Motif B region were mainly in the form of precipitation. After PPV epitope was chimeric in Motif A region, and the purified recombinant protein could be assembled into cVLPs. IFA results demonstrated that cVLPs retained a similar capacity to enter PK15 cells as its wild type. The formed cVLPs could stimulate the mouse body to produce antibodies against the PCV2 VLPs, the B5-E1 epitope and PPV VP2 protein, respectively. The results showed that Motif A in the Loop GH region of PCV2 Cap protein, can display foreign epitopes on the outer surface of virus-like particles, and can be recognized by immune cells to produce specific antibodies.

Key words: porcine circovirus type 2, virus-like particles, 3-fold axes region, foreign epitope

中图分类号:

S852.659.2

蔡云凤, 李宏, 黄小铭, 何庆, 丁振宇, 余婉婷, 罗施乐, 陈方志, 王乃东, 杨毅, 湛洋. 猪圆环病毒2型病毒样颗粒三倍轴区域嵌合猪细小病毒抗原表位的展示策略及免疫原性[J]. 畜牧兽医学报, 2025, 56(1): 307-318.

CAI Yunfeng, LI Hong, HUANG Xiaoming, HE Qing, DING Zhenyu, YU Wanting, LUO Shile, CHEN Fangzhi, WANG Naidong, YANG Yi, ZHAN Yang. Study on the Presentation Strategy and Immunogenicity of Porcine Parvovirus Epitope Inserted by 3-fold Axes Region on the Surface of Porcine Circovirus Type 2 Virus-like Particles[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 307-318.

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引物名称 Primer name	引物序列(5′→3′) Primer sequence (5′→3′)
PCV2-cap-F1	CGCCATATGCGGGGTTCTCATCATC
PCV2-cap-R1	CGCGGATCCTTATTTCGGATTCAGCGG
PCV2-GH-D1B-F2	TCACCCCGAAACCGTTTCAACCGAACAATAAGC
PCV2-GH-D1A-R2	GTTGAAACGGTTTCGGGGTGAAATAACG
PCV2-GH-D2B-F2	CTGTGGCTGCGCCTGCAACACGTGGGTCTGGGCACC
PCV2-GH-D2A-R2	CCCACGTGTTGCAGGCGCAGCCACAGCTGG
PCV2-GH-M1B-F2	CCG$\underline{{\rm{GCCGCAGCGGCCGCAGCGGCCGCG}}}$TTTCAACCGAACAATAAG
PCV2-GH-M1A-R2	GAAACG$\underline{{\rm{CGGCCGCTGCGGCCGCTGCGGC}}}$CGGTTTCGGGGTGAAA
PCV2-GH-M2B-F2	CAA$\underline{{\rm{GCCGCAGCGGCCGCAGCG}}}$CACGTGGGTCTGGGC
PCV2-GH-M2A-R2	GTG$\underline{{\rm{CGCTGCGGCCGCTGCGGCTTG}}}$CAGGCGCAGCCA
PCV2-GH-M3B-F2	CCG$\underline{{\rm{CGCCGTCGCCGTCGCCGTCGCCG}}}$TTTTCAACCGAACAAT
PCV2-GH-M3A-R2	GAAA$\underline{{\rm{ACGGCGACGGCGACGGCGACGGCG}}}$CGGTTTCGGGGTGAA
PCV2-GH-M4B-F2	CAAC$\underline{{\rm{GCCGTCGCCGTCGCCGTC}}}$ACGTGGGTCTGGGC
PCV2-GH-M4A-R2	GTG$\underline{{\rm{ACGGCGACGGCGACGGCG}}}$TTGCAGGCGCAGCCA
PCV2d-GH-I1B-F2	AACCG$\underline{{\rm{CAGCAGATTACCGATGCG}}}$TTTCAACCGAACAAT
PCV2d-GH-I1A-R2	GAAA$\underline{{\rm{CGCATCGGTAATCTGCTG}}}$CGGTTTCGGGGTGAAATAAC
PCV2d-GH-I2B-F2	CTGCAACAG$\underline{{\rm{CAGATTACCGATGCGCACGTGGGTCTGGG}}}$CACC
PCV2d-GH-I2A-R2	CACGTG$\underline{{\rm{CGCATCGGTAATCTGCTG}}}$TTGCAGGCGCAGCCAC

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猪圆环病毒2型病毒样颗粒三倍轴区域嵌合猪细小病毒抗原表位的展示策略及免疫原性

Study on the Presentation Strategy and Immunogenicity of Porcine Parvovirus Epitope Inserted by 3-fold Axes Region on the Surface of Porcine Circovirus Type 2 Virus-like Particles

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