表达猪瘟病毒 E2蛋白重组乳酸乳球菌的构建及其在小鼠中的免疫原性评估

Construction of recombinant Lactococcus lactis strains expressing classical swine fever virus E2 antigen and evaluation of their immunogenicity in murine models

摘要:

猪瘟病毒(classical swine fever virus, CSFV)主要通过呼吸道和消化道感染猪，给全球养猪业造成了巨大的经济损失。sIgA介导的黏膜免疫反应对于抵御CSFV早期入侵至关重要，本研究旨在利用乳酸乳球菌为载体研制口服疫苗，通过诱导黏膜免疫产生sIgA，从而阻断CSFV的早期入侵，为适应现代化猪场的大规模、无应激免疫提供新的技术手段。首先通过荧光素酶活体成像技术和粪便菌落计数评估乳酸乳球菌NZ9000在小鼠体内的定植能力，随后利用同源重组技术构建菌体表面展示CSFV E2蛋白的重组乳酸乳球菌NZ9000/pNZ8148- pgsA- E2以及胞内表达E2蛋白的重组乳酸乳球菌NZ9000/pNZ8148- E2。重组菌株经免疫印迹和间接免疫荧光鉴定目的蛋白E2的表达，随后评价重组菌株在小鼠体内的免疫原性。结果表明，乳酸乳球菌NZ9000可在小鼠体内短暂定植。NZ9000/pNZ8148- pgsA- E2与NZ9000/pNZ8148- E2分别实现了目的蛋白的表面展示与胞内表达。经灌胃免疫小鼠后，2种重组菌株均能有效诱导小鼠产生特异性免疫应答；表面展示型重组菌在诱导Th1型细胞因子分泌及CD4? T细胞活化方面比胞内表达型重组菌更具优势。本研究成功构建了表达猪瘟病毒E2蛋白的重组乳酸乳球菌，为开发防治猪瘟的黏膜免疫疫苗提供了实验依据。

Abstract:

Classical swine fever virus (CSFV) infects pigs primarily via the respiratory and digestive tracts and inflicts enormous economic losses on the global swine industry. To address the challenge of early CSFV invasion, this study developed an oral vaccine based on a Lactococcus lactis vector to induce sIgA-mediated mucosal immunity. This approach aims to provide robust early protection and a scalable, stress-free vaccination strategy for modern pig farming. In this study, we first assessed the colonization capacity of L. lactis NZ9000 in mice by in vivo luciferase imaging and fecal bacterial counting. We then employed homologous recombination to construct two recombinant L. lactis strains: NZ9000/pNZ8148- pgsA- E2 for surface display of the CSFV E2 protein and NZ9000/pNZ8148- E2 for intracellular expression of E2. The expression of E2 protein was confirmed by Western blot and indirect immunofluorescence assay. Immunogenicity of the recombinant strains was evaluated in mice. The results showed that L. lactis NZ9000 achieved short-term colonization in mice. NZ9000/pNZ8148- pgsA- E2 and NZ9000/pNZ8148- E2 achieved surface display and intracellular expression of E2, respectively. Both recombinant strains induced specific immune responses following gavage. Notably, NZ9000/pNZ8148- pgsA- E2 showed higher capacity in promoting Th1-type cytokine secretion and enhancing CD4 ⁺ T cell activation than NZ9000/pNZ8148- E2. This study successfully constructs recombinant L. lactis strains expressing the CSFV E2 protein and supports the further development of oral mucosal vaccines against classical swine fever.