ABSTRACT
Objective:To investigate the modulatory effect of IL-29 on trypsin-induced protease activated receptors (PARs) ex-pression on P815 mast cell.Methods:After P815 mast cells were challenged with different concentrations of IL-29 alone or combined with trypsin for 2 h, 6 h and 16 h, the challenged cells were collected and analysed by flow cytometry to detect the protein expression of PARs on P815 cells, and analysed by real time RT-PCR to detect the mRNA expression of PARs on P 815 cells.Results:Compared with the corresponding control , IL-29 induced significantly decreased expression of PAR-1 at protein and mRNA level on P815 cells, and upregulated PAR-3, PAR-4 mRNA level on P815 cells, whereas IL-29 did little effect on the expressions of PAR-2,3,4 at protein level on P815 cells accordingly.Preincubation of mast cell with IL-29 did not alter trypsin-induced PAR-1 expression on P815 cells, whereas up-regulated expression of PAR-2, 3, 4 were detected when P815 cell were pre-treated with IL-29 before being challenged with trypsin compared with the corresponding control .Conclusion: IL-29 can upregulate trypsin-induced PARs expression on mast cells through which participated in mast cell related inflammation .
ABSTRACT
Objective: To clone cDNA of human interleukin-29(IL-29) and express it in cos-7 cells, and to study its anti-HBV activity in vitro. Methods: Total RNA was extracted from PBMCs which had been infected with vesicular stomatitis virus(VSV) in vitro.IL-29 cDNA was amplified using one-step RT-PCR technique. The recombinant expressing plasimd of psectagB/His-myc-IL29 was constructed by inserting IL-29 cDNA into the vector and was then transfected into cos-7 cells. Stable expression stains were screened by Hygromycin B and limited dilution method. The target protein was purified through Ni2+-chelating Sepharose Fast Flow. Anti-viral bioactivity of the recombinant IL-29 fusion protein was analyzed through an in vitro model of production of HBV by the HepG2.2.2.15 cell line.ELISA was used for detection of the viral titers in the cell cultural supernants. Results: IL-29 was cloned and stably expressed in cos-7 cells successfully. SDS-PAGE and Western blot analysis showed multiple bands of the target protein with the molecular weights between 20 000 and 33 000, and the major band was located at about 33 000, indicating the fused IL-29 modified by additional glycosylation. The rhIL-29 was shown to dose-dependently inhibit secretion of HBsAg and HBeAg accompanied by the reduction of HBV genomic DNA in the cells tested. The inhibition ratio of HBsAg and HBeAg production was attained 85% at a concentration of 160 ?g/L of rhIL-29. Conclusion: The rhIL-29 with anti-HBV activity has been obtained.