| Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), also known as Blue-Ear Pig Desease, is a virus that causes a disease of pigs, called Porcine Reproductive and Respiratory Syndrome Respiratory Syndrome (PRRS). This economically important pandemic disease causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-strand RNA virus that belongs to the Arteriviridae family. PRRSV grows in primary alveolar macrophages and in monkey kidney cell lines. The genomic RNA is approximately 15 kb. The genome encodes the RNA replicase (ORF1a and ORF1b), the glycoproteins GP2 to E, the integral membrane protein M, and the nucleocapsid protein N (ORFs 2 to 7). Among them, ORF5 and ORF6 encode two major membrane-associated proteins, the envelope glycoprotein GP5 (E protein) and the nonglycosylated membrane protein M, respectively, which form disulfide-linked heterodimers (GP5/M) in the virus particle.The baculovirus expression vector system (BEVS) has become one of the most widely used systems for production of recombinant proteins, and has expressed a lot of proteins successfully. This system has advantages in expressing eukaryotic proteins, which cannot be expressed to bacterial expression systems, because the insect cells can post-translationally modify onto recombinant proteins similarly to their natural proteins. Recently, a bacmid (a baculovirus shuttle vector) system has been developed for BmNPV. The BmNPV bacmid can be replicated in Escherichia coli as a large plasmid, generates recombinmant virus DNA by site-specific transposition in E.coli, and remains infectious in insect cell. Because this method eliminates the need for multiple rounds of purification and amplication of virus, it markedly decreases the technical difficulty and the time required to select and purify recombinant virus. Because the manipulating is between the bacterial and baculovirus, we can name the technology as Bac-to-Bac system. The level of protein expression using the silkworm is 10-100 fold higher than that using B. mori cells, indicating that the silkworm is an optimal system for the mass production of recombinant proteins. From this point of view, BmNPV Bac-to-Bac using B. mori silkworm larvae is the most suitable combination for the large-scale production of eukaryotic proteins.In this paper, we expressed the E,M,E-2A-M proteins in the silkworm cell lines and silkworm larvae. It is suitable to use as molecular marker or fusion companion protein. We demonstrated a high-level expression of the E,M,E-2A-M gene in the Bombyx mori, silkworm larvae by liposome transfection. And then obtained virus from the hemolymph of the silkworm larvae where the E,M,E-2A-M were released with the cell lysated. It does not need to obtain virus from the cell culture. The virus can be stored as seed which could be used for the large-scale expression. Thus, for large-scale preparation of such recombinant virus, the Bac-To-Bac system using silkworm would be attractive due to low cost, being easy to treat, and having high safety for biohazard. At the same time, large-scale expression of the active foreign protein using silkworm larvae by the recombinant virus is easier to manipulate. Further investigation of E,M,E-2A-M protein should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.
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