重组SVA-PCV2二联疫苗的构建与免疫效果评价

doi:10.13560/j.cnki.biotech.bull.1985.2026-0228

摘要/Abstract

摘要：

目的研制一种能够同时针对猪圆环病毒2型（PCV2）与猪塞内卡病毒A（SVA）的高效二联疫苗，以应对当前生猪养殖中两种疫病混合感染与防控困难的问题。方法基于SVA反向遗传操作系统，通过同源重组方式将国内流行优势基因型PCV2d的结构蛋白Cap基因插入SVA基因组2A与2B基因之间，构建重组感染性克隆质粒pSVA-PCV2d-Cap。利用CMV启动子转录系统在BHK-21细胞中拯救重组病毒rSVA-PCV2d-Cap，并通过连续传代评估其遗传稳定性。采用间接免疫荧光、Western blot及PCR方法鉴定重组病毒中外源Cap蛋白的表达及其遗传稳定性。进一步将重组病毒灭活后与ISA206佐剂乳化制备灭活疫苗，免疫小鼠通过中和试验检测血清中抗SVA与抗PCV2特异性抗体水平。结果成功构建并拯救出可稳定表达PCV2d Cap蛋白的重组病毒rSVA-PCV2d-Cap，该病毒在PK-15细胞中传代10次后仍能保持Cap基因的完整性与表达稳定性。Western blot与间接免疫荧光结果显示，重组病毒感染细胞后可同时检测到SVA VP2蛋白与PCV2d Cap蛋白。小鼠免疫试验结果表明，重组病毒灭活疫苗免疫组可诱导产生抗SVA和抗PCV2的中和抗体的平均滴度分别为1∶128和1∶32，最高可达1∶256和1∶64。结论利用SVA载体系统可实现PCV2d Cap蛋白在哺乳动物细胞系中的稳定表达，所制备的重组病毒灭活疫苗能同时诱导针对SVA与PCV2的特异性免疫应答，为开发SVA-PCV2二联疫苗提供了可行的技术路径。

关键词: 猪圆环病毒2型, 猪塞内卡病毒A, 反向遗传操作系统, 重组病毒, SVA VP2蛋白, PCV2d Cap蛋白, 特异性免疫应答, PCV2-SVA二联疫苗

Abstract:

Objective To develop an efficient bivalent vaccine that simultaneously targets porcine circovirus type 2 (PCV2) and Senecavirus A (SVA), addressing the challenges of mixed infection and control and prevention of these two diseases in current swine farming. Method Using the SVA reverse genetics system, the structural protein Cap gene of the predominant domestic genotype PCV2d was inserted between the 2A and 2B genes of the SVA genome via homologous recombination to construct the recombinant infectious clone plasmid pSVA-PCV2d-Cap. The recombinant virus rSVA-PCV2d-Cap was rescued in BHK-21 cells using a CMV promoter-driven transcription system, and its genetic stability was evaluated through successive passages. Indirect immunofluorescence, Western blot, and PCR were employed to identify the expression of the exogenous Cap protein and its genetic stability in the recombinant virus. Furthermore, the recombinant virus was inactivated, emulsified with ISA206 adjuvant to prepare an inactivated vaccine, and used to immunize mice. Serum levels of anti-SVA and anti-PCV2 specific antibodies were detected via neutralization assays. Result The recombinant virus rSVA-PCV2d-Cap, capable of stably expressing the PCV2d Cap protein, was successfully constructed and rescued. The virus maintained the integrity and stable expression of the Cap gene after 10 consecutive passages in PK-15 cells. Western blot and indirect immunofluorescence results demonstrated the simultaneous detection of both SVA VP2 protein and PCV2d Cap protein in cells infected with the recombinant virus. The results of the mouse immunization experiment showed that the inactivated recombinant virus vaccine induced average neutralizing antibody titers of 1∶128 against SVA and 1∶32 against PCV2, with maximum titers reaching 1∶256 and 1∶64, respectively. Conclusion The SVA vector system can achieve the stable expression of the PCV2d Cap protein in mammalian cell lines. The prepared recombinant inactivated vaccine can concurrently induce specific immune responses against both SVA and PCV2, providing a feasible technical pathway for the development of a SVA-PCV2 bivalent vaccine.

Key words: porcine circovirus type 2, Senecavirus A, reverse genetics system, recombinant virus, SVA VP2 protein, PCV2d Cap protein, specific immune response, PCV2-SVA bivalent vaccine

李秀青, 王思宇, 刘懿文, 付士豪, 逯萍, 李昌瑶, 曾润帆, 郭运泽, 杨德成, 张晓战. 重组SVA-PCV2二联疫苗的构建与免疫效果评价[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0228.

LI Xiu-qing, WANG Si-yu, LIU Yi-wen, FU Shi-hao, LU Ping, LI Chang-yao, ZENG Run-fan, GUO Yun-ze, YANG De-cheng, ZHANG Xiao-zhan. Construction and Immunological Efficacy Evaluation of a Recombinant SVA-PCV2 Bivalent Vaccine[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0228.

图/表 5

图1 重组病毒质粒的构建1‒5表示5个单菌落培养物扩增产物

Fig. 1 Construction of recombinant viral plasmids1-5 indicate the amplification products from five single-colony cultures

图2 重组病毒rSVA-PCV2的拯救A：未转染的PK-15细胞；B：rSVA-PCV2d-Cap感染的PK-15细胞。标尺：400 μm

Fig. 2 Rescue of the recombinant virus rSVA-PCV2A: Untransfected PK-15 cells. B: PK-15 cells infected with rSVA-PCV2d-Cap. Scale bar: 400 μm

图3 重组病毒稳定性鉴定结果A：重组病毒P2-P10中Cap基因表达的PCR扩增（P2-P10表示重组病毒rSVA-PCV2d-Cap连续在PK-15细胞中传代次数）；B：重组病毒Cap蛋白表达的Western blot分析；C：重组病毒与亲本病毒Cap蛋白表达的Western blot比较分析

Fig. 3 Identification results of recombinant virus stabilityA: PCR amplification of Cap gene expression in recombinant virus passages P2-P10 (P2-P10 represent the passage numbers of recombinant virus rSVA-PCV2d-Cap serially passaged in PK-15 cells). B: Western blot analysis of Cap protein expression in recombinant virus. C: Western blot analysis comparing Cap protein expression between recombinant virus and parental virus

图4 重组病毒IFA鉴定结果（标尺400 μm）

Fig. 4 IFA identification results of the recombinant virus (Scale bar 400 μm)

图5 各组灭活疫苗免疫后产生PCV2和SVA中和抗体水平A：PCV2中和抗体检测；B：SVA中和抗体检测。***表示与对照组相比差异极显著（P<0.001）

Fig. 5 Neutralizing antibody levels against PCV2 and SVA in each group after immunization with inactivated vaccinesA: Detection of neutralizing antibodies against PCV2. B: Detection of neutralizing antibodies against SVA. *** indicates extremely significant difference compared with the control group (P<0.001). a. rSVA-PCV2d-Cap. b. PCV2/HeN-11/2019. c. SVA/HeN-1/2018. d. Control

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