To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes.
Amino Acid Sequence ; Base Sequence ; DNA ; genetics ; DNA Restriction Enzymes ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Plasmids ; Receptors, Polymeric Immunoglobulin ; genetics

BACKGROUND AND OBJECTIVES: The presence of eosinophil-specific cytotoxic mediators in nasopharyngeal secretions of infants with more severe RSV infection in the respiratory tract suggests that eosinophils play a key role in the pathogenesis of RSV-induced airway inflammation and the associated epithelial damage. A recent report demonstrated that RSV-infected respiratory cells induce eosinophil degranulation by a CD11b/CD18-dependent, ICAM-1-independent mechanism. However, the molecule on the epithelial cell membrane involved in the receptor-mediated degranulation of eosinophils after RSV infection has not been clearly identified. MATERIALS AND METHOD: We investigated the effect of RSV infection on the expression of pIgR on A549 cells and blocking of the RSV-infected cell induced eosinophil degranulation with monoclonal antibodies of the pIgR. RESULTS: After 24h of RSV infection, A549 cells expressed pIgR remarkably whereas pIgR was hardly expressed by the uninfected cells in flow cytometry and in the semi-quantitative RT-PCR. CD11b/CD18 on eosinophils was highly expressed by the RSV conditioned media. Purified eosinophils cocultured with the RSV-infected A549 cells showed approximately eightfold increase in ECP in the isotype control, compared with the control and that was blocked by treatment of anti-pIgR monoclonal antibody. CONCLUSION: It is strongly suggested that pIgR expression in the epithelial cells may be a key factor for eosinophil degranulation via interaction with CD11b/CD18 in the RSV-infected epithelial cells.
Antibodies, Monoclonal ; Culture Media, Conditioned ; Eosinophils* ; Epithelial Cells* ; Flow Cytometry ; Humans ; Infant ; Inflammation ; Membranes ; Polymers* ; Receptors, Polymeric Immunoglobulin* ; Respiratory Syncytial Viruses* ; Respiratory System