猫传染性腹膜炎病毒mRNA疫苗转录载体的构建与鉴定

doi:10.11843/j.issn.0366-6964.2025.02.029

摘要/Abstract

摘要：

本研究旨在设计猫传染性腹膜炎病毒(feline infectious peritonitis virus，FIPV)mRNA疫苗体外转录载体，并评估其转录出的FIPV mRNA疫苗的免疫原性。选取FIPV的核衣壳(nucleocapsid，N)蛋白，通过序列优化，结合CureVac mRNA技术平台，选择合适的非编码序列、信号肽序列和poly A尾部。将构建完成的目的序列克隆到pBluescript Ⅱ KS(+)载体上，经过载体的线性化单酶切后，在体外进行转录，合成编码FIPV N基因的mRNA，并经过加帽、纯化和琼脂糖凝胶电泳分析。通过体外转染试验验证抗原蛋白在细胞内的表达情况。在小鼠体内接种FIPV N-mRNA疫苗后，检测其体液免疫和细胞免疫反应。琼脂糖凝胶试验表明，设计的mRNA体外转录载体成功制备出稳定单一的mRNA序列。转染进细胞后，可在12~24 h内稳定表达目标抗原蛋白。ELISA试验结果显示FIPV N-mRNA疫苗在小鼠体内引起了强烈的体液免疫反应，其特异性抗体、IL-4、TNF-α水平明显高于对照组。Elispot试验结果显示FIPV N-mRNA组脾细胞分泌的IFN-γ的含量显著高于对照组。以FIPV N蛋白为抗原的体外转录载体所转录的mRNA疫苗，在细胞内能够高效表达目的蛋白，具有良好的免疫原性，有效引起小鼠的体液免疫和细胞免疫反应。FIPV N-mRNA疫苗有望成为猫传染性腹膜炎的潜在候选疫苗，mRNA体外转录载体的制备为传染性疾病mRNA疫苗的设计与研发提供了重要的参考价值。

关键词: 猫传染性腹膜炎, 猫传染性腹膜炎病毒N蛋白, mRNA疫苗, 体外转录载体

Abstract:

This study aimed to design an mRNA vaccine vector for feline infectious peritonitis virus (FIPV) and evaluate the immunogenicity of the transcribed FIPV mRNA vaccines. The nucleocapsid (N) protein of FIPV was selected, and its sequence was optimized, Using CureVac's mRNA technology platform, appropriate non-coding sequence, signal peptide sequences, and poly A tails were chosen. The synthesized target sequence was cloned into the pBluescript Ⅱ KS(+) vector, linearized by a single enzyme digestion, and transcribed in vitro to synthesize mRNA encoding the FIPV N gene. The transcribed mRNA sequence was capped, purified, and analyzed by agarose gel electrophoresis. In vitro transfection assays were used to verify the expression of antigen proteins in cells. Subsequently, mice were immunized with the FIPV N-mRNA vaccine and their humoral and cellular immune responses were assessed. Agarose gel electrophoresis confirmed the successful preparation of a stable single mRNA sequence using the designed mRNA transcription vector. After transfection into cells, the target antigen protein could be expressed stably within 12-24 hours. ELISA results showed a robust humoral immune response to the FIPV N-mRNA vaccine in mice, with significantly higher levels of specific antibodies, IL-4, and TNF-α compared to the control groups. Elispot assay results demonstrated significantly higher levels of IFN-γ secreted by spleen cells in the FIPV N-mRNA group compared to the control groups. The mRNA vaccine transcribed by the vector targeting the FIPV N protein efficiently expressrs the desired protein in cells and exhibits good immunogenicity, eliciting both humoral and cellular immune response in mice. The FIPV N-mRNA vaccine may be a preparation of mRNA transcription vector provides important reference value for the design and development of mRNA vaccines against infectious diseases.

Key words: feline infectious peritonitis virus, nucleocapsid protein, mRNA vaccines, in vitro transcription vectors

中图分类号:

R318

卢娜, 高钰, 赵嘉伟, 苏迪, 陈家磊, 罗忠礼. 猫传染性腹膜炎病毒mRNA疫苗转录载体的构建与鉴定[J]. 畜牧兽医学报, 2025, 56(2): 803-813.

LU Na, GAO Yu, ZHAO Jiawei, SU Di, CHEN Jialei, LUO Zhongli. Construction and Characterization of Transcription Vectors for Feline Infectious Peritonitis Virus mRNA Vaccines[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 803-813.

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引物名称 Primer name	序列(5′→3′) Sequence
EGFP-F	CCGGAATTCATGGTGAGCAAGGGCGAGGAGCTGTT
EGFP-R	CCCAAGCTTTTACTTGTACAGCTCGTCCATGCCG

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