Protective effects of bicistronic DNA vaccines of herpes simplex virus type 2 glycoprotein D and adjuvant CCL28 on mice / 中华微生物学和免疫学杂志

Objective:To study the protective effects of bicistronic DNA vaccines carrying herpes simplex virus type 2 glycoprotein D (HSV-2 gD) and adjuvant CCL28 sequences that were connected by internal ribosome entry site (IRES) sequence in mouse model.Methods:The recombinant DNA vaccines, pgD-IRES-CCL28 and pCCL28-IRES-gD, encoding HSV-2 gD and adjuvant CCL28 were constructed with IRES sequence. After verified by sequencing, they were intramuscularly injected twice into BALB/c mice. Serum samples and vaginal lavage fluids were collected regularly. Splenocytes, mesenteric lymph node cells and rectal mucosa tissues were separated and collected. The titers of antigen-specific antibodies in immunized mice were analyzed with end-point ELISA. In vitro neutralization assay was used to measure neutralizing antibody titers in serum and vaginal lavage fluid after vaccination and virus challenge. CCL28-responsive immune cells in splenocytes, mesenteric lymph node cells and rectal tissues were detected by chemotaxis experiment and immunohistochemical staining. The protective effects of the bicistronic DNA vaccines were evaluated by fluorescent quantitative PCR, weighing and disease severity assessment. Humoral and cellular immune responses induced by the bicistronic DNA vaccines and their efficacy in immunoprotection were analyzed by comparing with pgD+ pCCL28 group. Results:IgG titers in serum samples and IgA antibody titers in vaginal lavage fluids of mice immunized with pCCL28-IRES-gD were similar to those in pgD+ pCCL28 group. The neutralizing ability of antibodies, the number of rectal mucosal IgA+ plasma cells and CCL28-responsive immune cells in mucosal tissues were increased in pCCL28-IRES-gD group. Serum neutralizing antibodies were not produced immediately in the mice challenged with HSV-2, but no weight loss, disease symptoms or death was observed. However, pgD+ pcDNA3.1 and pgD-IRES-CCL28 were ineffective against HSV-2 infection in mice.Conclusions:The recombinant bicistronic DNA vaccine of pCCL28-IRES-gD could induce stronger mucosal immune response in mice and provide better protective effects.

Promotion of self-nucleic acid fragments on the assembly of foot-and-mouth disease virus-like particles / 生物工程学报

The special nucleic acid fragments, 5' untranslated region (5' UTR) and internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV), which interact with the capsid proteins, were selected as scaffolds to investigate the assembly efficiency of foot-and-mouth disease (FMD) virus-like particles (VLPs). The assembled product was characterized by evaluation of particle size, surface potential, gel retardation assay, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. The results confirmed that the 5' UTR and IRES of FMDV co-assembled with the FMD VLPs and facilitated the assembly efficiency of FMD-VLPs. It demonstrates that the assembly efficiency of 75S particles of VLPs-5'UTR was significantly higher than those of the VLPs (P<0.001) and VLPs-IRES group (P<0.01). Comparatively the assembly efficiency of 12S particles of VLPs-IRES was significantly higher than those of the VLPs (P<0.000 1) and VLPs-5'UTR (P<0.000 1). It showed that the 5' UTR represented more effective in facilitating the assembly of VLPs. This study proposes an optimized strategy for improving the assembly efficiency of VLPs for the development of VLPs vaccine.

Effect of ferric ammonium citrate on ROS-induced HCV IRES translation / 基础医学与临床

Objective To identify the activity of HCV IRES translation differences and identify the relationship between HCV IRES translation activity and ROS in different concentrations of ferric ammonium citrate ( FAC) in-duction.Methods 1 ) Expression plasmid pCI-Rluc-HCV IRES-Fluc was confirmed by endonuclease digestion as well as luciferase transient expression in Huh-7 cell;2) Controlled by dual-luciferase reporter assay, the differ-ent translation activity of HCV internal ribosomal entry site ( IRES ) was examined in a concentration of 50 μmol/L and 300μmol/L of FAC induction;ROS fluorescent staining method was used to detect the activity of ROS in Huh-7 cells, Western blot method was used to detect the protein expression changes of Nrf2 in Huh-7 cells;3) On the basis of the above experiments, 100 μmol/L DPI was added in 300 μmol/L FAC experimental group, to analyse the changes of HCV replication and ROS production after joining DPI.Results The generation of ROS and the activity of luciferase in the model group were significantly higher than that in the control group ( P<0.05 ) .FAC can enhance the expression of HCV IRES and increase the production of ROS , then causing Nrf2 expression in Huh-7 cell.However,after adding ROS inhibitor DPI, the above functions in Huh-7 cell were weakened.Conclusions The increase of HCV IRES expression induced by FAC is related to excessive ROS pro-duction induced by FAC in Huh-7 cells.

Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant.

Aim: To investigate if any mutations in hepatitis C virus (HCV) internal ribosome entry site (IRES) can inhibit the translation of viral polyprotein. Materials and Methods: A 26-year-old male patient infected with HCV 10 years ago was followed up. After 9 years of chronic infection. The patient had managed to resolve the infection for a period of 9 months, after which the patient experienced a viral recurrence characterized by high viral load and diverse HCV quasispecies. The IRES structures of the viral strains that disappeared were comparable with those that are currently active using structural mutational analysis. Results: A novo mutational position 254 combined with a rarely observed mutation at position 253 in the stem of the IIId subdomain were observed and the new conformation had an octa-apical loop (AGUGUUGG) and a shift in the 3 ` GU from the loop to the stem. Conclusions: These mutations were found to be highly deleterious, and they affected the direct binding of the IIId loop to the 40S ribosomal subunit with a subsequent inhibition of translation of viral polyprotein and clearance of the virus.

Generation of islet β cell-specific Cre recombinase targeting vector by homologous recombination in bacteria / 第二军医大学学报

Objective To construct a knock-in targeting vector for expressing of Cre recombinase specifically in islet β cell and provide the key material for knock-in mice with Crr recombinase expressed in islet β cells, providing a knock-out animal model for studying the function of islet β cells. Methods In our study, we constructed a knock-in targeting vector using the third exon of insulin 2 (Ins2) as a target site with λ phage Red recombination system. Through a first homologous recombination, we cloned an about 12 kb genomic DNA fragment from the bacterial artificial chromosome (BAC) which contained Ins2 genomic DNA into a low copy vector pBR322-2s through gap repair. Meantime, a mini-targeting vector containing internal ribosome entry site (.IRES), DNA sequences encoding Crr recombinase and positive-negative-selection (PNS) gene was generated. After second recombination, the final Ins2-Cre targeting vector was generated. Results With the third exon of Ins2 used as the target, we successfully constructed the knock-in targeting vector expressing Cre recombinase which was controlled by endogenous Ins2 gene. Conclusion We have successfully constructed the knock-in targeting vector expressing Cre recombinase, it will provide an important material forcreating animal model of Cre recombinase which is specially expressed in islet β cells.