ABSTRACT
This study was performed to investigate the effects of different regions of the Autographa califor nica multiple nucleopolyhedrovirus envelope protein E25 on its trafficking into nucleus and nuclear localization in host cells and on virus replication. Fourteen recombinant bacmids, each containing an e25 mutant with substitution or insertion of egfp, in the absence or presence of the native e25, were constructed and used to transfect Sf9 cells. The E25-EGFP fusion proteins and native E25 expressed in the cells transfect ed with individual recombinant bacmid were traced by autofluorescence from EGFP or by immuno-fluorescence assays. Confocal microscopy revealed that the E25-EGFP fusion protein with the N-domain (2-45aa) of E25 substituted by EGFP only distributed in the cytoplasm in transfected cells; and the fusion protein with EGFP inserted at the laa/2aa site of E25 completely remained outside of the nucleus and resided along the nuclear membrane. The E25-EGFPs with 46-118aa of E25 substituted by EGFP or with EGFP inserted at the 118aa/119aa site were present outside, across from the nuclear membrane or in nuclear plasm in dot-like shapes. The fusion proteins with the C-domain substituted by EGFP or with EGFP inserted at the site of 45/46aa or at the C-terminal formed a condensed ring or spread throughout the nucleus, in a similar manner to the E25 distributed in the cells transfected by the e25-knockout repair bacmid. These results prove that the N-terminal domain is critical for nuclear transportation of E25 and possibly to its position on the cytoplasm membrane as well; and the sequence downstream of the N-terminal domain also affects trafficking and nuclear localization of the protein. In cells transfected with bacmids containing both the native e25 and individual e25-egfp mutants, the E25-EGFP fusion proteins co-localized with E25 individually, showing similar patterns of subcellular localization as E25 mutants in the absence of native E25 in most cases, suggesting that the E25 likely exists and functions as dimmers or polymers. Production of infectious BV was dramatically reduced and even completely eliminated in most cases, either in the absence or presence of the native e25.
Subject(s)
Animals , Amino Acid Motifs , Cell Nucleus , Metabolism , Virology , Mutation , Nucleopolyhedroviruses , Chemistry , Genetics , Physiology , Protein Transport , Spodoptera , Virology , Viral Proteins , Chemistry , Genetics , Metabolism , Virus Release , Virus ReplicationABSTRACT
One alkaline protease from Actinomucor elegans AS3.2778 was purified protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.
ABSTRACT
One alkaline protease from Actinomucor elegans AS3.2778 was purified protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.
ABSTRACT
<p><b>OBJECTIVE</b>To study the specific cellular immune response induced by a recombinant HBV DNA vaccine and evaluate the potential therapeutic effect of this vaccine against HBV.</p><p><b>METHODS</b>A series of HBV epitopes were screened and the optimal combinations of these gene fragments identified to construct the target gene HS which incorporated appropriate flanking sequences. HS was inserted in pVAX1 to construct the recombinant plasmid PVAX-HS. Generation of specific cytotoxic T lymphocytes (CTLs) induced by PVAX-HS in HLA-A2 mice was observed by 51Cr assay and the activity of the CTLs evaluated using ELISPOT assay.</p><p><b>RESULTS</b>PVAX-HS was constructed successfully, which induced specific CTLs with strong activities after immunization in mice as compared with the control group that yielded negative results.</p><p><b>CONCLUSION</b>PVAX-HS DNA immunization can induced specific cellular immune response in mice, suggesting the potential therapeutic effect of this HBV vaccine.</p>
Subject(s)
Animals , Humans , Mice , Hepatitis B Vaccines , Genetics , Allergy and Immunology , Hepatitis B virus , Genetics , Allergy and Immunology , Immunization , Methods , Interferon-gamma , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Metabolism , Vaccines, DNA , Genetics , Allergy and ImmunologyABSTRACT
One factor at a time design and first order exponential model were applied to investigate the effects of the four major factors(temperature,ionic strength,pH and osmo-regulator) in the preservation process of nitrite oxidization bacteria(NOB),and a first order decay kinetics was introduced.The result showed that the anoxic decay rate of nitrifying bacteria reduced from 0.25 to 0.015,the half life was extend to 53d at 4℃,pH7.60,ionic strength 0.035mol/kg.The protecting effect of glycerin showed obviously better than that of other cryoprotectants at 4℃.