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Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines
Protein Expression and Purification ( IF 1.6 ) Pub Date : 2020-09-21 , DOI: 10.1016/j.pep.2020.105763
Sochanwattey Meas , Phenjun Mekvichitsaeng , Yaowaluck Maprang Roshorm

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.



中文翻译:

通过双顺反子和三顺反子DNA疫苗共表达源自猪繁殖与呼吸综合征病毒的自我切割的多种蛋白

猪繁殖与呼吸综合症病毒(PRRSV)引起的猪繁殖与呼吸综合症仍然是养猪业中的重要疾病之一。需要一种安全,有效并且还引发针对多种抗原的广泛免疫应答的疫苗。在这项研究中,我们开发了能够共表达源自PRRSV的多种结构蛋白的多顺反子DNA疫苗。为了保留每种抗原蛋白的结构和功能,我们采用了自切割2A肽来介导由多顺反子基因表达的多种蛋白的分离。生成了六个编码PRRSV GP5和M蛋白的双顺反子基因,每个构建体包含源自口蹄疫病毒(F2A),猪破伤风病毒1(P2A)和Thatasigna的不同2A序列。病毒(T2A)具有或不具有弗林蛋白酶切割位点(Fu)。在哺乳动物表达质粒pTH的载体下,所有六个双顺反子基因以可比较的水平共表达蛋白质GP5和M。重要的是,所有六种类型的2A序列都可以介导GP5和M的完全自我切割。我们接下来生成了共表达PRRSV蛋白GP5,M和N的三顺反子DNA疫苗。P2A和F2A的所有同源和异源组合三顺反子基因中的α-内酰胺酶产生GP5,M和N蛋白的完全自我切割。我们的研究报告了从一种疫苗以离散形式共表达多种PRRSV结构蛋白的成功,并证实了开发一种可提供针对PRRSV的广泛免疫应答的单一疫苗的可行性。

更新日期:2020-09-26
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