PMID: 18724696Aug 30, 2008Paper

Construction of adenoviral vector encoding soluble human sTNFRI-IgGFc cDNA and its expression in human airway smooth muscle cells (HASMCs)

Sheng wu gong cheng xue bao = Chinese journal of biotechnology
Jin SuYong-ta Huang

Abstract

Tumour necrosis factor (TNF-a) is a pro-inflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma, and which has recently been highlighted as potentially important in refractory asthma. To study the feasibility of local treatment of asthma with recombinant adenovirus vector carrying soluble extra-cellular region of TNF receptor I-IgGFc (sTNFRI-IgGFc) fusion protein, The sTNFRI-IgGFc gene was subcloned into the adenovirus shuttle plasmid pDC316, the products were co-transfected into HEK293 cell line with helper plasmid pBHGloxDeltaE1,3Cre. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was produced by homologous recombination of above 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, its titer was measured by TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in transfected human airway smooth muscle cells (HASMCs) was detected by RT-PCR, ELISA and immunological histochemistry. The anti-TNF activity assay of transfected HASMCs culture supernatant was measured by MTT. Ad-sTNFRI-IgGFc was successfully constructed with the titer of 3x10(10) TCID50/mL. Ad-sTNFRI-IgGFc can transfect HASMC with high efficacy. The transcription of...Continue Reading

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