Establishment of a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8] / 中国实验动物学报

Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus(RV) G1P[8] infection to these cells,and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8].Methods The primary small intestinal epithelial cells were obtained by collagenase Ⅺ and dispase I digestion from tree shrew.After purification and identification,the obtained primary small intestinal epithelial cells were infected with RV.Then,culture supernatants of infected cells were collected every 12 hours after infection.Viral titer and viral load were subsequently determined.Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells.The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew,high purity above 90% primary tree shrew small intestinal epithelial cells was obtained.These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells,HCT116 cells and MA104 cells.The virus titer reached to 2.0×105TCID 50/mL at 72 h after infection.Using Western blot and indirect immunofluorescence observation,the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells,and were located in the cytoplasm from days 1 to 5.Conclusions The separation,purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful,and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells / 中国病毒学

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein)genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.