基于融合 PCR 构建猪瘟病毒反向遗传操作系统

Construction of reverse genetic system of classical swine fever virus based on fusion PCR

摘要

[目的]本文旨在建立猪瘟病毒(classical swine fever virus, CSFV)Shimen 株的反向遗传操作系统,为 CSFV 新型疫苗研制和致病机制研究提供技术平台。[方法]根据 CSFV Shimen 株的基因序列设计了 4 对引物,利用这 4 对引物扩增出 4 段涵盖 CSFV 基因组全长的 DNA 片段,并在第一段的 5′端引入 T7 启动子序列,随后通过同源重组的方法将 4 个片段分别克隆至低拷贝载体 pWSK-29 中。以构建的低拷贝质粒为模板,采用融合 PCR 方法,扩增出病毒基因组全长 DNA 序列,经体外转录并转染 PK-15 细胞。拯救的病毒分别经培养上清液传代和带毒细胞传代的方式进行繁殖。[结果]带毒细胞传代方式可获得更高滴度的病毒。间接免疫荧光试验结果显示,拯救病毒能够在 PK-15 细胞中产生特异性荧光信号。Western blot 检测证实拯救病毒能够表达 CSFV 特异性蛋白。病毒生长曲线分析表明,拯救病毒与亲本病毒具有相似的增殖特性,均在接种后 48~60 h 达到病毒滴度高峰。[结论]成功建立了 CSFV Shimen 株的反向遗传操作系统,为后续 CSFV 新型疫苗的研制、基因功能研究以及致病机制的深入探索奠定了重要基础。

Abstract

[Objectives] The paper aimed to establish a reverse genetic system for the CSFV Shimen strain, providing a technical platform for the development of novel CSFV vaccines and the research into its pathogenic mechanisms. [Methods] Based on the genomic sequence of the CSFV Shimen strain, four pairs of primers were designed to amplify four DNA fragments that span the full length of the CSFV genome and T7 promoter sequence was introduced into the 5' end of the first segment. Subsequently, the four fragments were cloned into the low-copy vector pWSK-29 by homologous recombination. The full-length DNA sequence of the viral genome was amplified by fusion PCR using the constructed low-copy plasmids as templates, followed by in vitro transcription and transfection into PK-15 cells. The rescued virus was propagated using both standard virus passage (conventional viral propagation) and infected-cell passage. [Results] The experimental results confirmed that the infected-cell passage method yielded a higher viral titer. IFA results demonstrated that the rescued virus produced specific fluorescence signals in PK-15 cells. Western blot confirmed that the rescued virus expressed CSFV-specific proteins. Virus growth curve analysis indicated that the rescued virus exhibited proliferation characteristics similar to those of the parental virus, with both reaching peak titers at 48-60 h. Using these methods, a reverse genetic system for the CSFV Shimen strain was successfully established. [Conclusions] This study successfully established a reverse genetic system for the CSFV Shimen strain, providing a foundation for the development of novel CSFV vaccines, gene function studies, and further exploration of its pathogenic mechanisms. The establishment of this system will not only facilitate further studies of CSFV molecular biology but also offer valuable insights into the reverse genetic manipulation of other viruses in the Pestivirus genus.

关键词

猪瘟病毒 / 反向遗传技术 / 同源重组 / 融合 PCR

Key words

CSFV / reverse genetic technology / homologous recombination / fusion PCR

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基金资助

国家自然科学基金项目(32172840)