High Expression Of RSV G And RSV M Drosophila Expression Vectors In S2Cells

Human respiratory syncytial virus (RSV) is an enveloped non-segmented negative-sense RNA virus, which is classified into Family Paramyxoviridae. The RSV is now identified as the most important viral agent of lower respiratory tract illness (LRI) in infants and a significant problem in elderly, persons with cardiopulmonary diseases, and immunocompromised hosts worldwidely. The RSV genome is15.2kb in length and is transcribed into10separate major mRNAs that encode11identified proteins. Attachment protein (G) is one of the main neutralized antigens for RSV, while Matrix protein is presumed to play a crucial role in virus assembly, budding and the formation of virus particles. Getting large amounts of high quality protein is a primary problem for diagnostic reagent and vaccines.At present, Bac-to-Bac baculovirus expression vector system is the most commonly used for the RSV protein expression. Yet, it is tedious and difficult to purify the proteins. The Drosophila expression system is a simple and well characterized system that combines some of the advantages of the mammalian and baculovirus expression.In this study, we use the Drosophila expression system to construct four plasmids of RSV G and RSV M, high expressing RSV G and RSV M Drosophila Expression Vectors in S2Cells The target gene including signal peptide is successfully amplified by PCR and then transformed into T vector. Plasmids were extracted and sent for sequencing. After digested by restriction enzyme, the target gene was inserted into pMT/V5-His A vector successfully. The resultant plasmids were cotransfected with pCoHygro, respectively, by using calcium phosphate transfection method. After removing calcium phosphate, hygromycin B of300μg/ml was used to screen the transfected cells. The culture medium was refreshed every3-5days. After induced by500μM CuSO4for48h, the target protein expression is confirmed by Western blot.In this research we construct four plasmids:pMT-SG-His, pMT-SQ pMT-SM and pMT-SM-His strains of stable S2cells expressing SG-His, the SG, the SM and SM-His are obtained.