猪繁殖与呼吸综合征病毒GP5和M蛋白的真核表达及免疫原性评价

Eukaryotic expression of GP5 and M protein of porcine reproductive and respiratory syndrome virus and immunogenicity evaluation

摘要:

为了解我国猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)的流行及进化情况并开发针对流行谱系的亚单位疫苗，对2001-2021年间分离于我国的PRRSV毒株进行遗传进化分析，选用优势流行谱系代表毒株，优化其GP5和M蛋白的基因序列，与干扰素(interferon, IFN)和免疫球蛋白的Fc区串联后，构建真核表达质粒pCDNA3.4-IFNα-GP5-Fc和pCDNA3.4-IFNα-M-Fc，并使用HEK293T真核表达系统表达重组蛋白IFNα-GP5-Fc和IFNα-M-Fc。将2种重组蛋白与ISA206VG佐剂混匀，免疫断奶仔猪，通过酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)及中和试验评价体液免疫水平，酶联免疫斑点试验(enzyme-linked immunospot assay, ELISPOT)评价细胞免疫水平。试验结果表明，NADC30-like谱系为近年我国主要流行的谱系，IFNα-GP5-Fc和IFNα-M-Fc组合免疫仔猪能诱导高水平的抗体和细胞免疫，该研究为制备更为安全有效的新型PRRSV亚单位疫苗奠定基础。

关键词:猪繁殖与呼吸综合征(PRRS);遗传进化分析;GP5重组蛋白;M重组蛋白;免疫原性评价

Abstract:

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.

Key words:porcine reproductive and respiratory syndrome virus (PRRS);genetic evolution analysis;GP5 recombinant protein;M recombinant protein;immunogenicity evaluation