The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.
Monoclonal antibodies (MAbs) against human recombinant interferon beta (hrIFNbeta) were generated by genetic immunization (GI). In order to test two viral promoters frequently used in mammalian expression plasmid vectors, mice were inoculated four times by intramuscular injection, without adjuvant, with 100 microg of either pcDNA 3.1hrIFNbeta or pZeoSV2IFNbeta containing the entire human interferon beta gene and under the control of, respectively, human cytomegalovirus (HCMV) immediate-early promoter or early SV-40 enhancer/promoter. Only serum samples from mice immunized with pZeoSV2IFNbeta were positive to anti-hrIFNbeta. The spleens of the immunized mice were fused with myeloma Sp2/0 cells and the hybridoma clones generated screened by an in house enzyme-linked immunosorbent assay (ELISA). Fourteen MAbs were selected as reactive with hrIFNbeta. Western blot analysis was performed and only one recognized the 18 kDa isoform (non-glycosylated) of hrIFNbeta. All MAbs were subjected to antibody isotype characterization with a commercial ELISA and showed unusual profile with simultaneous expression of both IgM and IgG2a isotypes. This observation is further supported by RT-PCR amplification of the IgM CH4 domain using total RNA from hybridomas.
The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.
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