ABSTRACT
<p><b>OBJECTIVE</b>To explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice.</p><p><b>METHODS</b>As model antigen, the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4.0 with intron A involved and pVAX1 without intron A involved, respectively. The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines. Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining.</p><p><b>RESULTS</b>Compared with non-intron A pVAX1hsp65, the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAX1hsp65 caused higher expression level of Hsp65 in 293T cells, and enhanced Th1 type immune response, which was defined as higher level of anti-Hsp65 specific total IgG level (3.76 ± 0.23 vs 3.15 ± 0.22, P < 0.01) and IgG2a/IgG1 ratio (4.08 ± 0.04 vs 2.23 ± 0.12, P < 0.01) and more IFN-γ-secreting CD4(+) ((2.0 ± 0.058)% vs (1.5 ± 0.087)%, t = 4.804, P < 0.01) and CD8(+) ((0.6 ± 0.058)% vs (1.0 ± 0.115)%, t = 3.098, P < 0.05) T lymphocytes. The difference showed statistical significance.</p><p><b>CONCLUSION</b>Intron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.</p>
Subject(s)
Animals , Female , Mice , Bacterial Proteins , Genetics , Allergy and Immunology , Chaperonin 60 , Genetics , Allergy and Immunology , Genetic Vectors , Introns , Allergy and Immunology , Mice, Inbred BALB C , Plasmids , Vaccines, DNA , Genetics , Allergy and ImmunologyABSTRACT
Antimicrobial peptides are a class of small peptides with anti-extrogenous pathogen activities.They are derived from organism and possess antibacterial,antifungus,antiviruses and anticancer cell actions.In recent years,it’s found that some microbial pathogens are able to resist antimicrobial peptides.The constitutive and inducible mechanism of a pathogen resists a given peptide were reviewed in this paper.
ABSTRACT
The TSR1(thermosensitive rescued)gene associated with the secretion of proteins of the yeast Yarrowia lipolytica was site-directed mutagenesis,restricted and spliced to obtain a series of deletion mutants.These mutants would be useful for studing the function of different domains of TSR1 gene in yeast Yarrowia lipolytica
ABSTRACT
A novel nucleic acid amplification method,termed loop-mediated isothermal amplification(LAMP),which amplifies DNA with high specificity,efficiency,and rapidity under isothermal conditions,may be a valuable tool for the rapid detection of infectious diseases.This method employs a DNA polymerase that have activity of strand displacement DNA synthesis and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.LAMP can amplify a few copies of DNA to 109 in less than an hour.The final products are stem-loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops.A positive reaction would be shown as a ladder-like pattern in a gel electrophoresis analysis.Because of the advantage,the LAMP method will be widely applied to research of nucleic acid,clinical diagnosis of infectious diseases and detection of genetically modified organisms etc.