ABSTRACT
<p><b>OBJECTIVE</b>To analyze the influence of optimal codon usage on the expression levels and immunogenicity of DNA vaccines, encoding the human papillomavirus type 6b (HPV 6b) E7 gene.</p><p><b>METHODS</b>The full length E7 gene of HPV 6b was modified to substitute human preferred codon for rarely used codon, and three mutations were introduced into the pRB binding site of HPV 6b E7 to eliminate its transformation potential. The codon optimized and mutated E7 gene (hu-mE7) were cloned into the Kpn I and EcoR I site of the pcDNA3 mammalian expression vector, the in vitro expression of the hu-mE7 gene and the immunogenicity of hu-mE7 DNA vaccine were compared with the wt-E7gene.</p><p><b>RESULTS</b>The in vitro expression of pcDNA3-hu-mE7 was much higher than the classical wt-E7 plasmid in monkey COS-1 cell line. Mice immunized intramuscularly with the pcDNA3-hu-mE7 showed that the codon modified E7 gene induced a stronger IFN-gamma ratios than the wt-E7 gene.</p><p><b>CONCLUSIONS</b>These results suggest that the optimized codon usage contributes to the enhancement of gene expression and immunogenicity of HPV 6b E7 gene.</p>
Subject(s)
Animals , Female , Mice , Cell Line , Codon , Genetics , Gene Expression , Genes, Viral , Genetics , Genetic Vectors , Papillomaviridae , Genetics , Papillomavirus Vaccines , Transfection , Vaccines, DNA , Allergy and Immunology , Viral Proteins , Genetics , Viral Vaccines , Allergy and ImmunologyABSTRACT
AIM: To elucidate molecular mechanisms underlying calcium handling protein in diastolic heart failure (DHF) from mRNA level and protein expression, including calcium adenosine triphosphatase (Ca 2+ -ATPase), phospholamban, ryanodine receptor, calsequestrin and L-type calcium channel. METHODS: The mRNA of these calcium handling genes were detected by RT-PCR, and the protein levels were analyzed by Western blot analysis. RESULTS: Compared with sham-operated rabbits, the steady-state levels of mRNA encoding the SR Ca 2+ -ATPase and cardiac L-type calcium channel were decreased significantly in rabbits with DHF, and protein level of SR Ca 2+ - ATPase was greatly reduced, whereas the mRNA and protein levels of other calcium handling protein were unchanged. CONCLUSION: L-type calcium channel and the sarcoplasmic reticular Ca 2+ -ATPase were down regulated in DHF. These changes may be a contributory factor for DHF.