ABSTRACT
Objective: To construct, express and purify ScFvl4/EGFP fusion proteins which containing Arg9, and to study their binding activities and internalization functions. Methods: Arg9 gene was recombined into 5' terminal, 3' terminal of ScFv/EGFP gene and between them respectively before they were cloned into the expression vector pET32a. After induced in E. coli BL21 and purified, their binding activities and internalization were respectively analyzed by indirect ELISA and indirect immunofluorescence analysis. Results: DNA sequencing and restriction endonuclease digestion proved that the four fusion genes were correctly constructed. SDS-PAGE analysis and Western blot showed that they were successfully expressed and purified. Indirect ELISA confirmed that the expressed products had antigen specific binding activities. Indirect immunofluorescence analysis revealed the fusion protein containing Arg9 at its N terminal had much better internalization function, but never internalized into the cells which do not express HBsAg. Conclusion: The four fusion genes were constructed, expressed and purified successfully. The purified fusion proteins maintained the binding activities to HBsAg and the fusion protein containing Arg9 at its N terminal had much better internalization effect.
ABSTRACT
<p><b>OBJECTIVE</b>To observe the inhibitory effect of targeted ribonuclease delivered via adenovirus against HBV replication in vitro.</p><p><b>METHODS</b>The shuttle plasmids pDC316 were constructed on the basis of the previous plasmids pcDNA3.1(-)/TRL, pcDNA3.1(-)/TR, pcDNA3.1(-)/TRmut, pcDNA3.1(-)/HBVc, and pcDNA3.1(-)/hEDN by subcloning the target gene sequences of TRL, TR, HBVc, and hEDN, respectively. HEK 293 cells were cotransfected with the pDC316 plasmids respectively in the presence of the rescue plasmid pBHGlox(delta)E1,3Cre to yield the recombinant adenoviral vectors which comprised the above genes. After transfection of HepG2.2.15 cells with the vectors, RAd/TRL expression was detected by indirect immunofluorescence staining and RT-PCR. Radioimmunoassay was used to analyse anti-HBV activity of RAd/TRL.</p><p><b>RESULTS</b>Recombinant RAd vectors were prepared successfully. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of HBsAg and HBeAg concentration in comparison with the controls.</p><p><b>CONCLUSION</b>Adenoviral vector-mediated targeted ribonuclease can effectively inhibit HBV replication.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Cell Line , Cell Line, Tumor , Fluorescent Antibody Technique, Indirect , Gene Expression , Genetic Vectors , Genetics , Hepatitis B Core Antigens , Genetics , Metabolism , Hepatitis B virus , Genetics , Metabolism , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleases , Genetics , Metabolism , Transfection , Virus Replication , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To adapt the candidate strains of hemorrhagic fever with renal syndrome (HFRS) purified vaccine to Vero cells and to study their antigenicity and immunogenicity.</p><p><b>METHODS</b>The viral strains H8207 (Hantaan virus, HTN) and Y86013 (Seoul virus, SEO) were continuously propagated in Vero cell by the terminal dilution method and studied the characteristics of virus multiplication, viral titers and the amounts of virus antigen after serial passages. Three batches of crude monovalent inactivated vaccine were developed using the different passages of these 2 viral strains.</p><p><b>RESULTS</b>The strains H8207 and Y86013 adapted to Vero cells and stably grew on the cells with high titers. Rabbits immunized with the crude vaccines of H8207 and Y86013 showed 100% sero-conversion and the neutralizing antibody titers of the rabbit immune sera reached 1?10 at 4 weeks after 2 times of immunization.</p><p><b>CONCLUSIONS</b>The results suggest that these 2 candidate strains had adapted to Vero cells, possessed high titers and good immunogenicity and be feasible to prepare the HFRS purified vaccine in Vero cells.</p>
Subject(s)
Animals , Mice , Rabbits , Antibodies, Viral , Blood , Chlorocebus aethiops , Hantaan virus , Allergy and Immunology , Hemorrhagic Fever with Renal Syndrome , Neutralization Tests , Seoul virus , Allergy and Immunology , Vaccination , Vaccines, Inactivated , Allergy and Immunology , Vero Cells , Viral Vaccines , Allergy and ImmunologyABSTRACT
<p><b>AIM</b>To observe the expression of estrogen receptors alpha and beta in human tongue squamous cancer line Tca8113 cell, and to study the influence of beta-estradiol (beta-E2) on the proliferation and cell cycle of cultured Tca8113 cell.</p><p><b>METHODS</b>Immunocytochemistry and RT-PCR methods were used to observe the expression of estrogen receptors (ER) in human tongue squamous carcinoma line Tca8113 cell. 3H-TdR incorporation and cell cycle analysis were used to examine the change of proliferation and DNA synthesis of Tca8113 cell.</p><p><b>RESULTS</b>ER-alpha and ER-beta mRNA were expressed in human tongue squamous cancer cell, and the expression of ER-beta was weaker than that of ER-alpha. beta-Estradiol at 10(-8) mol/L - 10(-6) mol/L could increase the proliferation of human tongue squamous carcinoma cell in a dose dependent manner (P < 0.01). beta-E2 (10(-6) mol/L) could increase the proportion of cells in S phase and G2 phase from 23.5% up to 37.7%. The effect of estradiol on the proliferation of cultured human tongue squamous cancer line Tca8113 cell could be inhibited by Tamoxifen.</p><p><b>CONCLUSION</b>There are ER-alpha and ER-beta expression in human tongue squamous cancer line Tca8113 cell, and beta-estradiol promotes the proliferation and cell cycle of cultured human Tca8113 cell.</p>