非洲猪瘟病毒p30蛋白与CRM197的融合表达及对小鼠的免疫原性评价

doi:10.11843/j.issn.0366-6964.2024.11.038

摘要/Abstract

摘要：

非洲猪瘟是由非洲猪瘟病毒引起的一种急性、高传染性和致死性的传染病，对猪养殖业造成了巨大的影响。安全有效的疫苗和有效治疗药物的缺乏给非洲猪瘟防控带来了巨大的挑战。作者尝试设计一种融合蛋白以改善非洲猪瘟病毒p30的免疫原性。白喉毒素的无毒突变体CRM197已被成功地用作多糖-蛋白结合疫苗的载体蛋白，以增强多糖的免疫原性。本研究构建p30与CRM197催化结构域(A片段)的融合表达质粒，纯化后的重组蛋白和单独p30分别使用小鼠评价了其免疫原性。结果显示：通过原核表达与亲和层析纯化得到融合蛋白p30-CRM197(A)免疫小鼠后，较单独表达的p30其可以在更短的时间内激发起高水平针对p30的特异性IgG，并且在初次免疫后56 d后还能维持较高水平的淋巴细胞免疫活力。本研究结果表明，CRM197的A片段作为载体蛋白显著增强原核表达p30蛋白的免疫原性，其可作为非洲猪瘟亚单位疫苗的候选分子内佐剂。

关键词: 非洲猪瘟, CRM197, p30, 免疫原性

Abstract:

African swine fever is an acute, highly contagious and deadly infectious disease caused by the African swine fever virus, which has had a huge impact on the pig breeding industry. The lack of vaccines and effective therapeutic drugs has brought great challenges to the prevention and control of African swine fever. We tried to design a fusion protein to improve the immunogenicity of African swine fever virus p30. CRM197, a non-virulent mutant of diphtheria toxin, has been successfully used as a carrier protein in polysaccharide-protein conjugate vaccines to enhance the immunogenicity of polysaccharides. In this study, a fusion expression plasmid of p30 and CRM197 catalytic domain (A fragment) was constructed, and the immunogenicity of the purified recombinant protein and p30 alone were evaluated using mice. After immunizing mice with the fusion protein p30-CRM197(A) purified by prokaryotic expression and affinity chromatography, p30 alone could excite a high level of specific IgG against p30 in a shorter period of time, and maintain a high level of lymphocyte immune activity two months after the initial epidemic. The results of this study showed that the A fragment of CRM197 alone could significantly enhance the immunogenicity of prokaryotic expression p30 protein as a protein carrier, and it could be used as a candidate intramolecular adjuvant for African swine fever subunit vaccine.

Key words: African swine fever, CRM197, p30, immunogenicity

中图分类号:

S852.659.1

张琦, 董宁宁, 谈晓梅, 石正旺, 李娜, 朱雯琪, 汤傲星, 李传锋, 朱杰, 刘光清, 苏艳, 孟春春. 非洲猪瘟病毒p30蛋白与CRM197的融合表达及对小鼠的免疫原性评价[J]. 畜牧兽医学报, 2024, 55(11): 5230-5237.

Qi ZHANG, Ningning DONG, Xiaomei TAN, Zhengwang SHI, Na LI, Wenqi ZHU, Aoxing TANG, Chuanfeng LI, Jie ZHU, Guangqing LIU, Yan SU, Chunchun MENG. Fusion Expression of African Swine Fever Virus p30 Protein and CRM197 and Evaluation of Its Immunogenicity in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5230-5237.

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目的基因 Target gene	寡核苷酸序列(5′→3′) Oligonucleotide sequence
p30	F: GCCATGGCTGATATCGGATCCATGAAAATGGAGGTCATC
p30	R: TCCACCACCACCAGATCCACCACCACCTTTTTTTTTTAAAAGTTTAATAAC
CRM197(A)	F: GGTGGTGGATCTGGTGGTGGTGGATCTATGGGCGCGGATGATGTG
CRM197(A)	R: CTCGAGTGCGGCCGCAAGCTTGCGCACGCGGTTGCCCGC

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