ABSTRACT
@#Objective To express recombinant human interleukin-29-Fc(rhIL-29-Fc) fusion protein in human embryonic kidney 293-F(HEK293F) cells and analyze its anti-tumor activity in vitro.Methods The recombinant expression plasmid UCOE-IL-29-Fc was constructed and transiently transfected into HEK-293F cells.After expression and purification,rhIL-29-Fc fusion protein was obtained and identified by SDS-PAGE and Western blot;Female Japanese white rabbits were immunized with rhIL-29 and rhIL-29-Fc protein subcutaneously in the left ear respectively,2 rabbits in each group,0.5 mg per rabbit.Blood samples were collected from the vein of right ear,and the serum was separated.The half-life was measured by ELISA and the anti-proliferation effect of rhIL-29-Fc protein on human colon cancer HT-29,human colon cancer HCT-116,human Burkkit lymphoma Daudi,human non-small cell lung cancer NCI-H1975,human small cell lung cancer NCI-H209,human esophageal cancer EC109 and human pancreatic cancer PANC-1 cells in vitro was detected by CCK-8 assay,and the inhibitory concentration 50(IC_(50)) was calculated.Results The recombinant expression plasmid UCOE-IL-29-Fc was constructed correctly as identified by double digestion and sequencing.After transient transfection into HEK-293 cells for 6 d,the culture supernatant was harvested.The relative molecular mass of the purified rhIL-29-Fc fusion protein was consistent with the expectation.The protein showed a specific binding reaction with mouse anti-human IL-29 monoclonal antibody with a concentration of 1.5 mg/ml and a purity of 93%.RhIL-29-Fc protein had a half-life of 25 h and showed different inhibitory effects on the proliferation of 7 kinds of tumor cells,and the IC_(50) on different cells was also different.Conclusion The rhIL-29-Fc fusion protein was successfully expressed in HEK-293F cells,and the half-life of the fusion protein was 20 h longer than that of rhIL-29.According to the different anti-tumor proliferation activity in vitro and IC_(50) results on 7 kinds of tumor cells,it was found that the anti-tumor activity of rhIL-29-Fc fusion protein was higher than that of rhIL-29.This study laid a foundation of the development of IL-29 protein in the treatment of tumors.
ABSTRACT
Objective To investigate the value of IL-27,IL-29 and miRNA-497 in HER-2 positive radioactive particles combined targeted therapy. Methods Sixty-one elderly inpatients with HER-2 positive gastric cancer ascites in the Gaoming District People's Hospital, Wuchuan Municipal People's Hospital and Zhejiang Provincial Tumor Hospital were divided into the two groups. The treatment group(n= 31) was treated by the radioactive particles+ targeted drug Trastuzumab(initial dose 8 mg/kg, maintenance dose 66 mg/kg, once per 3 weeks) for 6 weeks; the control group(30 cases) was treated by targeted drug Trastuzumab (initial dose 8 mg/kg,maintenance dose 6 mg/kg, once per 3 weeks) for 6 weeks. The levels of ascites IL-27,IL-29 and miRNA-497 in the radioactive partcles combined targeted therapy group and control group were detected. Results The levels of ascites IL-27, IL-29 and miRNA-497 after treatment in the treatment group were significantly decreased compared with those before treatment and in the control group,the effective remission rate in the treatment group was 74. 19%, which was higher than 36.67% in the control group, the average median survival time in the treatment group was 155 d, which washigher than 72 d in the control group, the difference was statistically significant. Conclusion IL-27 ,IL-29 and miRNA-497 has the significance of curative effect evaluation and prognosis judgment in the radioactive particles combined targeted therapy for the patients with HER 2 positive gastric cance.
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
BACKGROUND: Members belonging to the interferon-lambda (IFN-lambda) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-lambda biology, such as endocytosis of IFN-lambda, we produced monoclonal antibodies (Abs) against human IFN-lambda and examined their usefulness. METHODS: We purified recombinant human IFN-lambda1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-lambda1- immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-lambda1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-lambda and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. RESULTS: Four hybridoma clones secreting Abs specific to IFN-lambda1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-lambda1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-lambda1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-lambda complex on HepG2 cells. CONCLUSION: Monoclonal Abs against IFN-lambda1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-lambda.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Biology , Clone Cells , Endocytosis , Escherichia coli , Fluorescence , Hep G2 Cells , Hybridomas , RhodaminesABSTRACT
BACKGROUND: Members belonging to the interferon-lambda (IFN-lambda) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-lambda biology, such as endocytosis of IFN-lambda, we produced monoclonal antibodies (Abs) against human IFN-lambda and examined their usefulness. METHODS: We purified recombinant human IFN-lambda1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-lambda1- immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-lambda1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-lambda and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. RESULTS: Four hybridoma clones secreting Abs specific to IFN-lambda1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-lambda1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-lambda1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-lambda complex on HepG2 cells. CONCLUSION: Monoclonal Abs against IFN-lambda1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-lambda.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Biology , Clone Cells , Endocytosis , Escherichia coli , Fluorescence , Hep G2 Cells , Hybridomas , RhodaminesABSTRACT
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.