表达增强型绿色荧光蛋白的报告猪瘟病毒C株的构建及在家兔中生物学特性评价

doi:10.11843/j.issn.0366-6964.2020.07.022

摘要/Abstract

摘要： 猪瘟兔化弱毒疫苗C株是我国自主研发的，用于预防猪瘟（CSF）的最好弱毒活疫苗。然而，现阶段的C株不具备标记功能，无法区分野毒感染与疫苗接种。本研究旨在将增强型绿色荧光蛋白（EGFP）编码基因插入猪瘟病毒（CSFV）C株将其构建为报告病毒，为C株的基础研究提供病毒示踪工具，同时提供构建标记C株疫苗的方法。本研究中，笔者在CSFV C株中引入EGFP基因，分别构建了表达增强型绿色荧光蛋白（EGFP）的报告CSFV rHCLV-EGFP，以及用CSFV强毒Shimen（SM）株N^pro基因替换C株N^pro基因后再引入EGFP基因的嵌合报告病毒rHCLV-N^pro（SM）-EGFP。通过猪瘟抗原ELISA检测，直接观察荧光及Western blot检测EGFP的蛋白表达等方法对拯救的病毒进行鉴定，并在细胞和家兔上评价这两株病毒的生物学特性。结果显示，两株报告病毒的抗原ELISA结果均为阳性；在感染rHCLV-EGFP的细胞中并未观察到绿色荧光也未检测到EGFP蛋白，但在感染rHCLV-N^pro（SM）-EGFP的细胞中观察到EGFP的绿色荧光并检测到EGFP蛋白；细胞试验结果表明，两株报告病毒与亲本病毒具有相似的生长特性且遗传稳定；家兔试验发现，rHCLV-N^pro（SM）-EGFP保留了C株的在家兔体内的生物学特性，但rHCLV-EGFP失去了C株诱导家兔定型热反应的能力。构建的嵌合报告病毒rHCLV-N^pro（SM）-EGFP可以作为C株的报告病毒进行应用：可以对病毒示踪，用于研究C株的复制过程、病毒与细胞的相互作用；同时，该嵌合报告病毒也具备发展为标记C株疫苗的潜力。

关键词: 猪瘟病毒, C株, N^pro蛋白, 增强型绿色荧光蛋白, 生物学特性

Abstract: C-strain (also known as HCLV strain) of classical swine fever virus (CSFV) is a lapinized live attenuated vaccine against classical swine fever (CSF). However, C-strain lacks the capacity for the serological differentiation between infected and vaccinated animals (DIVA). The purpose of this study is to insert the gene encoding the enhanced green fluorescent protein (EGFP) into CSFV C-strain to construct a reporter virus, and to provide a virus tracing tool for basic research of C-strain, and also to provide a method for constructing C-strain marker vaccine. Here, we constructed and characterized two reporter viruses based on C-strain. One is the reporter virus rHCLV-EGFP, which expressing the enhanced green fluorescent protein (EGFP) fused in frame with the N^pro protein. The other one is the chimeric reporter virus rHCLV-N^pro(SM)-EGFP in which the N^pro gene is replaced with the counterpart of the highly virulent CSFV Shimen (SM) strain in the context of C-strain. The researchers identified the rescued reporter viruses by ELISA kit for CSFV antigen, observing the fluorescence directly and detecting the EGFP protein by Western blot, as well as evaluated the biological characteristics of these two reporter viruses in cells and rabbits. The antigens of the two reporter viruses were positive by ELISA. The reporter virus rHCLV-EGFP remained viable but not fluorescent, containing an intact EGFP gene but expressed as an N^pro-EGFP fusion protein with unexpected size. Interestingly, the EGFP fluorescence was restored in the chimeric reporter virus rHCLV-N^pro(SM)-EGFP. The experiment in cells showed that the two reporter viruses had similar growth characteristics to the parental virus and retained genetic stability. Furthermore, the animal experiment in rabbits revealed that rHCLV-N^pro(SM)-EGFP exhibited similar biological properties to the parental virus C-strain, while rHCLV-EGFP lost the ability to induce the fever response of C-strain in rabbits. In this study, the chimeric reporter virus rHCLV-N^pro (SM)-EGFP is able to be used as the reporter virus of C-strain. It can be used for virus tracing to study the replication process of the C-strain and the interaction between the virus and cells. The chimeric reporter virus also has the potential for a marker vaccine, which has the DIVA capability by detecting the anti-EGFP antibody.

Key words: classical swine fever virus, C-strain, N^pro protein, enhanced green fluorescent protein, characterization

中图分类号:

S852.651

韩玉莹, 谢利豹, 李永锋, 仇华吉. 表达增强型绿色荧光蛋白的报告猪瘟病毒C株的构建及在家兔中生物学特性评价[J]. 畜牧兽医学报, 2020, 51(7): 1699-1709.

HAN Yuying, XIE Libao, LI Yongfeng, QIU Huaji. Generation and Characterization in Rabbits of a Reporter Classical Swine Fever Virus Vaccine C-strain Expressing the Enhanced Green Fluorescent Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1699-1709.

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