[Construction of adenoviral vector encoding soluble human sTNFRI-IgGFc cDNA and its expression in human airway smooth muscle cells (HASMCs)].
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 sTNFRI-IgGFc mRNA and the expression of protein were confirmed in the transfected HASMCs. Moreover, the product in 100 microL expression supernatant could completely antagonize the cytolytic effect of 2ng TNFa on L929 cells, even at 1/64 dilution. This study forms the basement of the experiment study on local treatment of asthma with adenovirus expressing sTNFRI-IgGFc.
Su
Jin
J
Department of Respiratory Diseases, Nanfang hospital, Southern Medical University, Guangzhou 510515, China.
You
Changxuan
C
Cai
Shaoxi
S
Ma
Li
L
Wang
Xiaoning
X
Wen
Qian
Q
Luo
Wei
W
Huang
Yongta
Y
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D001249:Asthma / Q000628:therapy; D001980:Bronchi / Q000166:cytology; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha