表达猪圆环病毒3型Cap蛋白重组伪狂犬病病毒的构建及其对小鼠免疫效果评价

doi:10.11843/j.issn.0366-6964.2025.11.035

摘要/Abstract

摘要：

为构建表达猪圆环病毒3型(PCV3)Cap蛋白的重组病毒载体疫苗，以猪伪狂犬病病毒(PRV)SX1910毒株为亲本毒株，利用CRISPR/Cas9和同源重组技术构建了缺失gE和gI基因并携带LoxP位点和GFP荧光标记的重组病毒SX1910-ΔgE/gI-GFP。然后利用Cre酶将SX1910-ΔgE/gI-GFP的GFP表达盒置换为双拷贝的Cap基因表达盒，获得重组病毒rSX1910-ΔgE/gI-2Cap_PCV3，并对其进行体外生物学特性和免疫效果分析。结果表明，rSX1910-ΔgE/gI-2Cap_PCV3体外增殖能力和噬斑大小显著小于亲本毒株SX1910。将rSX1910-ΔgE/gI-2Cap_PCV3免疫接种至6周龄雌性BALB/c小鼠进行免疫效果初步评价，结果显示，rSX1910-ΔgE/gI-2Cap_PCV3可有效刺激小鼠产生Cap蛋白的抗体，有效减轻PCV3感染对肺部组织的损伤，显著降低组织病毒载量。以上数据表明，rSX1910-ΔgE/gI-2Cap_PCV3可对小鼠产生良好的免疫效果，并为PCV3新型疫苗的研究奠定了科学基础。

关键词: 猪圆环病毒3型, Cap蛋白, 猪伪狂犬病病毒, CRISPR Cas9, 免疫保护

Abstract:

This study aimed to construct a viral vector vaccine expressing the Cap protein of porcine circovirus type 3 (PCV3). In this study, pseudorabies virus (PRV) SX1910 strain was designated as the parental strain. Using CRISPR/Cas9 and homologous recombination technologies, a recombinant virus SX1910-ΔgE/gI-GFP was constructed, which lacked the gE and gI genes and harbored a LoxP site and GFP fluorescent marker. Then, the GFP expression cassette of SX1910-ΔgE/gI-GFP was replaced with the double expression cassette of the PCV3 Cap gene by using Cre enzyme, generating the recombinant virus rSX1910-ΔgE/gI-2Cap_PCV3. Then its in vitro biological characteristics and immunological effects were analysed. The results showed that rSX1910-ΔgE/gI-2Cap_PCV3 exhibited significantly reduced in vitro replication capacity and plaque size compared to the parental strain SX1910. Preliminary evaluation of immunogenicity in 6-week-old female BALB/c mice demonstrated that rSX1910-ΔgE/gI-2Cap_PCV3 effectively stimulated the production of Cap protein-specific antibodies in mice, alleviated PCV3-induced lung tissue damage, and significantly reduced viral loads in tissues. Therefore, rSX1910-ΔgE/gI-2Cap_PCV3 can elicit a good immune response in mice, providing a scientific foundation for the development of novel vaccines for PCV3.

Key words: porcine circovirus type 3, Cap protein, pseudorabies virus, CRISPR Cas9, immune protection

中图分类号:

S852.659.1

宋若琪, 谭姗姗, 马瑞一, 陈新新, 宜越, 马梦瑶, 王颖, 牛胜, 闫芳, 赵宇军, 田文霞, 任建乐. 表达猪圆环病毒3型Cap蛋白重组伪狂犬病病毒的构建及其对小鼠免疫效果评价[J]. 畜牧兽医学报, 2025, 56(11): 5778-5788.

SONG Ruoqi, TAN Shanshan, MA Ruiyi, CHEN Xinxin, YI Yue, MA Mengyao, WANG Ying, NIU Sheng, YAN Fang, ZHAO Yujun, TIAN Wenxia, REN Jianle. Construction and Immune Effect in Mice of Recombinant Pseudorabies Virus Expressing Porcine Circovirus Type 3 Cap Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5778-5788.

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引物名称 Primer name	序列(5′→3′) Sequence (5′→3′)	酶切位点	产物大小/bp Product/bp	用途 purpose
PCV3-3.1-F/R	F: CTAGCGTTTAAACTTAAGCTTATGAGACACAGA GCTATATT	Hind Ⅲ	687	扩增Cap基因
PCV3-3.1-F/R	R: AACGGGCCCTCTAGACTCGAGTTAGAGAACG GACTTGTAAC	XhoⅠ	687	扩增Cap基因
Cap3-SK1-F/R	F: CTATAGGGCGAATTGGGTACCGACATTGATTA TTGACTAGT	KpnⅠ	1 637	扩增Cap表达盒1
Cap3-SK1-F/R	R: TCCCCCGGGCTGCAGGAATTCCCATAGAGC CCACCGCATCC	EcoRⅠ	1 637	扩增Cap表达盒1
Cap3-SK2-F/R	F: TTCCTGCAGCCCGGGGGATCCGACATTGATTA TTGACTAGT	BamHⅠ	1 639	扩增Cap表达盒2
Cap3-SK2-F/R	R: AGCTCCACCGCGGTGGCGGCCGCCCATAGAG CCCACCGCATCC	Not Ⅰ	1 639	扩增Cap表达盒2
2Cap_PCV3-F/R	F: ATAACTTCGTATAGCATACATTATACGAAGTTATC TCACTATAGGGCGAATTGGGTACCGACATTGA		3 262	扩增和鉴定双拷贝的Cap基因表达
2Cap_PCV3-F/R	R: ATAACTTCGTATAATGTATGCTATACGAAGTTAT GAGCTCCACCGCGGTGGCGGCCGCCCATAGAG		3 262	扩增和鉴定双拷贝的Cap基因表达
QPCV3-F/R	F: CACGGAGGTCTGTAGGGAGA R: GGACACTCGTAGCACCACAA		156	荧光定量

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