稳定表达GM-CSF的重组塞内卡病毒A的构建与鉴定

Construction and identification of a recombinant Senecavirus A stably expressing porcine GM-CSF

塞内卡病毒A(Senecavirus A,SVA)是近年来新发现的一种能够引起猪水疱性相关疾病的病毒,该病毒在我国流行范围越来越广,对养猪业造成了巨大经济损失。为提高灭活疫苗的免疫效果,本研究以SVA感染性克隆为基础,将猪源GM-CSF-T2A基因通过融合PCR方法插入到SVA的2A和2B之间,成功构建了重组质粒SVA-GM-CSF,将其转染BHK-21细胞进行病毒拯救及传代。结果显示,重组病毒感染BHK-21细胞可以引起明显的细胞病变;经RT-PCR扩增与基因测序结果表明目的基因正确插入;间接免疫荧光鉴定表明外源基因GM-CSF在该病毒中成功获得了表达。生物学特性分析表明,重组病毒与亲本病毒在BHK-21细胞中具有相似的生长特性,并且该重组病毒在传代过程中具有良好的遗传稳定性。本研究已成功构建了稳定表达GM-CSF的重组SVA,该重组病毒的构建为进一步明确SVA致病机制和研制新型疫苗提供了理论支持与技术指导。

Senecavirus A(SVA),a newly emerged virus in recent years,it can cause swine vesicular disease,the virus has become more and more widespread in China,causing huge economic losses to the pig industry.Conventional inactivated SVA vaccine developed in some laboratories can effectively prevent and control the disease,but there are some defects such as low antibody level and short immune period.In order to improve the immune effect of inactivated vaccine,this study based on SVA infectious clone,and the porcine GM-CSF-T2A gene was inserted between 2A and 2B of SVA by fusion PCR,and the recombinant plasmid SVA-GM-CSF was successfully constructed,then it was transfected into BHK-21 cells for viral rescue and passage.The results showed that the recombinant virus in BHK-21 cells could cause obvious cytopathic effect.RT-PCR amplification and gene sequencing showed that the target gene was inserted correctly.Indirect immunofluorescence showed that the foreign gene GM-CSF was successfully expressed in this virus.Biological characterization analysis showed that the recombinant virus and the parent virus had similar growth characteristics in BHK-21 cells,and the recombinant virus had good genetic stability during the passage,the construction of the recombinant virus provides theoretical support and technical guidance for further clarifying the pathogenesis of SVA and developing a new vaccine.