ABSTRACT
To clone porcine bone marrow stromal antigen-2 (BST-2) gene, construct its recombinant eukaryotic expression plasmid and induce the expression of the fusion antiviral protein, we amplified BST-2 gene by RT-PCR from the total RNA extracted from PK15 cells. The recombinant expression plasmid pcDNA-BST-2 was constructed and then was transfected into HEK293T cells to expresse the BST-2 fusion protein. Western blot and indirect immunofluorescence assay (IFA) were performed, and the biological activity was detected. The results showed that the construction of recombinant plasmid pcDNA-BST-2 was confirmed by restriction enzyme digestion and sequencing. The expressed product had antiviral activity against Vesicular stomatitis virus (VSV), Avian influenza virus (AIV) and Porcine reproductive and respiratory syndrome virus (PRRSV). In conclusion, the research paves the way for further research on bioactivity assayand antiviral medication.
Subject(s)
Animals , Humans , Antigens, CD , Genetics , Allergy and Immunology , Cell Line , Chickens , Cloning, Molecular , Gene Expression , Influenza in Birds , Allergy and Immunology , Virology , Orthomyxoviridae , Physiology , Porcine Reproductive and Respiratory Syndrome , Allergy and Immunology , Virology , Porcine respiratory and reproductive syndrome virus , Physiology , Swine , Vesicular Stomatitis , Allergy and Immunology , Virology , Vesicular stomatitis Indiana virus , Physiology , Virus ReplicationABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of the cadmium chloride on the DNA damage and the expression of the gadd153 and gadd45beta promoter and mRNA in HepG2 cells.</p><p><b>METHODS</b>DNA damage induced by cadmium chloride was detected by comet assay. The plasmids (pGADD153-Luc and pG45-Luc) containing DNA damage and repair inducible gene 153 and 45 (gadd153 and gadd45beta) promoter and luciferase and gadd45beta reporter gene were constructed. The activity of gadd153 and gadd45beta promoter were represented by the luciferase activity, the inducible luciferase activities was detected by bioluminescence. The expression of gadd153 and gadd45beta mRNA was detected by RT-PCR.</p><p><b>RESULTS</b>The results of comet assay indicated that Olive Tail Moment induced by the cadmium chloride increased significantly at the dose of 100, 300 micromol/L, compared with the control (P < 0.05). The luciferase activity analysis showed that the expression levels of gadd153 promoter increased significantly in 1, 5, 10 micromol/L treatment group, compared with the control (P < 0.05). The expression levels of gadd45beta promoter in 5, 10 micromol/L treatment group were significantly higher than that in control group (P < 0.05). The expression levels of gadd153 mRNA induced by cadmium chloride at the doses of 1, 5, 10 micromol/L and the expression levels of gadd45beta mRNA induced at the doses of 5, 10 micromol/L were significantly higher than thoae in control group (P < 0.05).</p><p><b>CONCLUSION</b>The cadmium chloride can induce the DNA damage and increase the expression levels of the gadd153 and gadd45beta promoters in HepG2 cells.</p>