Construction and identification of recombinant replication-defective adenoviruses harbouring expression cassettes of I540-PTD and I540-PTD4
AIM: To construct the recombinant replication-defective adenoviruses harbouring the expression cassettes of I540-PTD and I540-PTD4 fusion genes. METHODS: Recombinant adenovirus was constructed by co-transfecting HEK293 cells with pDC315-I540-PTD and pDC315-I540-PTD4 with genomic plasmid pBHGloxdeltaE13Cre respectively. The infectious titers of Ad-I540-PTD and Ad-I540-PTD4 were tittered by end-point dilution assay and plaque assay. After infected with the recombinant viruses,the expression of I540-PTD and I540-PTD4 fusion proteins was analyzed by immunohistochemical staining. RESULTS: Ten days after co-transfecting HEK293 cells with pDC315-I540-PTD and pDC315-I540-PTD4 with genomic plasmid pBHGloxdel-taE13Cre respectively,typical cytopathic effects were seen. The lysates containing recombinant replication-defective adenoviruses Ad-I540-PTD and Ad-I540-PTD4 were obtained by three consecutive free-thaw cycles after harvesting the HEK293 cells. The infectious titers of Ad-I540-PTD and Ad-1540-PTD4 were (8. 9 × 1012)and (6.3 × 1012)pfu/L,(5.8 × 1012) and (5.4 × 1012) pfu/L determined respectively by end-point dilution assay and by plaque assay. Ad-I540-PTD and Ad-I540-PTD4 were confirmed by amplifying the 167 bp products by PCR with specific primers using recombinant adenoviruses DNA as templates. Immunohisto- chemical analysis using anti-His tag monoclonal antibody demonstrated that the U20S infected with Ad-I540-PTD and Ad-I540-PTD4 was both strongly positively stained. CONCLUSION: We have successfully designed and constructed the recombinant replication-defective adenoviruses Ad-I540-PTD and Ad-I540-PTD4 harbouring I540-PTD and I540-PTD4 expression cassette respectively.
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万方数据
维普
