]AIM: To construct a recombinant adenovirus shuttle plasmid pDC315 - SPA( gene promoter of pulmonary surfactant - associated protein A ) - mCLCA3 ( mouse calcium - activated chloride channel 3 ) and to study the targeting expression of mCLCA3 in airway epithelial cells.METHODS: The full length of SPA - mCLCA3 gene was cloned by PCR from PGL - SPA and pCR - Blunt II - TOPO vectors, and subcloned into pDC315 - EGFP to construct pDC315 - SPA - mCLCA3 targeting expression vector.After identified by nucleotide sequencing analysis, the vector pDC315 - SPA -mCLCA3 and control vector pDC315 - EGFP ( used for negative control as well as GFP positive control ) were transfected into H441 cells and HEK293 cells with lipofectamine 2000.The mRNA expression of mCLCA3 in each cell line was determined by RT - PCR.The protein level of mCLCA3 in transfected cells was detected by immunocytochemistry.RESULTS: The recombinant plasmid containing SPA promoter and mCLCA3 fusion gene was confirmed by sequencing analysis.The expression of mCLCA3 mRNA was undetectable in the 2 cell lines transfected with pDC315 - EGFP.In the cells transfected with pDC315 - SPA - mCLCA3, mRNA expression of mCLCA3 was significantly higher in H441 cells than that in HEK293 cell line ( P ＜0.05 ), indicating pDC315 - SPA - mCLCA3 targeting expression vector had high transcriptional targeting activity in airway epithelial cells.The result of immunohistochemistry showed that the protein expression of mCLCA3 was not detected in the control cells while strong immunoreactivity was observed in H441 cells transfected with pDC315 - SPA -mCLCA3.The protein expression of mCLCA3 was not detected in HEK293 cells with or without pDC315 - SPA - mCLCA3 transfection.CONCLUSION: The recombinant adenovirus shuttle plasmid pDC315 - SPA - mCLCA3 has the targeting expression specificity to airway epithelial cells.
Pulmonary surfactant - associated protein A promoterCLCA3 protein, mouseAirway epithelial cellsTargeting expression
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