Construction of foot-and-mouth disease virus like particles-induced expression vectors and screening of BHK-21 cell pools / 生物工程学报

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.

Establishment of SAV stablely expressed cell pool and purification of SAV interacting protein complex / 中国药学杂志

OBJECTIVE: To Establish HEK293 cell model in which SAV is stably expressing and purification of SAV interacting protein complex. METHODS: RT-PCR was used to amplify SAV gene segment in HEK293 cells and then the PCR product was cloned into pBabe-SBP-FLAG vector with BamH I and EcoR I restriction enzyme cutting sites. Co-transfection of pBabe-SBP-FLAG-SAV and the packing plasmids into HEK293T cells to produce retroviruses which will be used for infection of HEK293 cells. Stable cell pool was selected by puromycine for 2 weeks and SAV expression was detected by Western-blot. SAV interacting protein complex was purified by streptavidin beads from the stable cell pool and virulized by silver staining. RESULTS: pBabe-SBP-FLAG-SAV expression vector was successfully constructed. FLAG-SBP tagged SAV in HEK293 stable cell pool was detected by straight Western-blot and IP-Western-blot. SAV interacting protein complex was captured by streptavidin beads and some specific bands purified from SAV stable cell pool was visualized compare to control in silver stainning. CONCLUSION: pBabe-SBP-FLAG-SAV eukaryotic expression plasmid and the stable cell pool is successfully constructed and the interacting protein complex is purified by streptavidin beads, which provide a foundation for further investigation of SAV.

The Kinetics of Secondary Response of Antigen-Specific CD4+ T Cells Primed in vitro with Antigen

BACKGROUND: Memory T lymphocytes of the immune system provide long-term protection in response to bacterial or viral infections/immunization. Ag concentration has also been postulated to be important in determining whether T cell differentiation favors effector versus memory cell development. In the present study we hypothesized that na?ve Ag-specific CD4+ T cells briefly stimulated with different Ag doses at the primary exposure could affect establishment of memory cell pool after secondary immunization. METHODS: To assess this hypothesis, the response kinetics of DO11.10 TCR CD4+ T cells primed with different Ag doses in vitro was measured after adoptive transfer to naive BALB/c mice. RESULTS: Maximum expansion was shown in cells primarily stimulated with high doses of ovalbumin peptide (OVA323-339), whereas cells in vitro stimulated with low dose were expanded slightly after in vivo secondary exposure. However, the cells primed with low OVA323-339 peptide dose showed least contraction and established higher number of memory cells than other treated groups. When the cell division was analyzed after adoptive transfer, the high dose Ag-stimulated donor cells have undergone seven rounds of cell division at 3 days post-adoptive transfer. However, there was very few division in naive and low dose of peptide-treated group. CONCLUSION: These results suggest that primary stimulation with a low dose of Ag leads to better memory CD4+ T cell generation after secondary immunization. Therefore, these facts imply that optimally primed CD4+ T cells is necessary to support effective memory pool following administration of booster dose in prime-boost vaccination.