摘要
A recombinant subunit vaccine against Seneca Valley virus (SVV) would be valuable to the swine industry. Recent SVV outbreaks have raised concerns with national biosecurity. This is due to the potential of foreign vesicular animal diseases entering the United States undetected because of confounding clinical signs of SVV. Research with the baculovirus expression vector system (BEVS) has produced virus-like particles (VLP) that elicited neutralizing antibodies and protected against challenge for many Picornaviridae viruses. Building on this previous research, attempts of SVV VLP assembly for a vaccine candidate were performed using the BEVS.
All baculovirus constructs were designed to encode the SVV P1 region, 2A protease, portions of the 2B and 3B genes and 3C protease. Recombinant SVV proteins VP1, VP2 and VP3 were expressed in several baculovirus construct iterations. Peptide specific antibodies detected each protein by means of Western blot analysis. Sucrose gradient fractionation and electron microscopy (EM) were performed to verify VLP formation. However, VLP production was not confirmed by either method.
Several factors influence the assembly of SVV VLPs. One outcome providing insight of the complete cleavage of the recombinant capsid proteins is the identification of a 55kDa protein band. This band was detected in the α-SVV VP1 and α-SVV VP3 Western blots. Also, the 3C protease and its impact in the BEVS need further investigation to determine its role in complete cleavage of the recombinant capsid proteins. Lastly, there were indications the recombinant capsid proteins were aggregating instead of folding properly into VLPs. The gaps in information noted here support the need for further research into capsid formation in Picornaviridae viruses to improve VLP assembly using the BEVS.
摘要译文
针对塞内卡谷地病毒（SVV）的重组亚单位疫苗对养猪业将是有价值的。最近爆发的SVV引起了人们对国家生物安全的关注。这是由于由于SVV的临床症状混杂而导致未发现的外国水疱动物疾病进入美国的可能性。用杆状病毒表达载体系统（BEVS）进行的研究已经产生了病毒样颗粒（VLP），该颗粒引起中和抗体并能抵抗许多Picornaviridae病毒的攻击。在此先前研究的基础上，使用BEVS进行了针对候选疫苗的SVV VLP组装的尝试。设计所有杆状病毒构建体，以编码SVV P1区，2A蛋白酶，2B和3B基因的一部分以及3C蛋白酶。重组SVV蛋白VP1，VP2和VP3在几次杆状病毒构建体迭代中表达。肽特异性抗体通过蛋白质印迹分析检测到每种蛋白质。蔗糖梯度分级分离和电子显微镜（EM）验证了VLP的形成。但是，两种方法均未确认VLP的产生。几个因素会影响SVV VLP的组装。提供了对重组衣壳蛋白的完全切割的见解的一个结果是鉴定了55kDa蛋白带。在α-SVVVP1和α-SVVVP3 Western印迹中检测到该条带。同样，需要进一步研究3C蛋白酶及其在BEVS中的作用，以确定其在重组衣壳蛋白的完全切割中的作用。最后，有迹象表明重组衣壳蛋白正在聚集，而不是正确折叠成VLP。此处指出的信息空白表明，需要进一步研究细叶病毒科衣壳的形成，以改善使用BEVS的VLP装配。
English, Jennifer Lynn. Evaluation of Methods for Generating Senecavirus A Virus-Like Particles Utilizing the Baculovirus Expression Vector System[D]. US: Iowa State University, 2018