Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.
Active Transport, Cell Nucleus ; Animals ; Coronavirus Infections ; immunology ; veterinary ; virology ; Genes, Viral ; Host-Pathogen Interactions ; immunology ; Interferons ; antagonists & inhibitors ; biosynthesis ; genetics ; Interleukins ; antagonists & inhibitors ; biosynthesis ; genetics ; NF-kappa B ; metabolism ; Nucleocapsid Proteins ; genetics ; immunology ; physiology ; Porcine epidemic diarrhea virus ; genetics ; pathogenicity ; physiology ; Promoter Regions, Genetic ; Swine ; Swine Diseases ; immunology ; virology

Bactericidal/permeability-increasing protein (BPI) can bind to and specifically neutralize lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria. In order to evaluate potent LPS-neutralizing activity of bovine BPI, the full-length coding sequence (1 449bp) or 714 bp N-terminal coding sequence (BPI714) of bovine BPI was transfected into mHEK293 cells and the expression of LPS-induced inflammatory cytokines was studied. First, we constructed the lentiviral expression vectors and generated mHEK293 cells stably expressing recombinant bovine BPI or BPI714. Then, we detected the expression of IL-8, IL-1β, TNF-α, NF-κB-1 and NF-κB-2 genes by real-time PCR at 0, 1, 3, 6, 12, 24, 36 and 48 h post of LPS induction in cells with or without recombinant bovine BPI or BPI714 ectopic expression, respectively. In response to LPS, the robust abundance of inflammatory cytokines including IL-8, IL-1β, TNF-α and NF-κB-2 was observed in wild type mHEK293 cells at eachtime point. On the contrary, mRNA abundance of IL-8, TNF-α and NF-κB-2 in transfected mHEK293 cells showed no significant changes at each indicated time point. Our results demonstrated that recombinant bovine full length BPI or BPI714 down-regulated the expression of inflammatory cytokines and revealed that either of bovine BPI or BPI714 was able to inhibit the immune respond stimulated by LPS. This study provides evidence for further investigating the mechanisms and application of BPI/LPS-neutralizing activity and also documents a reliable approach for analysis of the efficacy of antibacterial proteins.
Animals ; Antimicrobial Cationic Peptides ; chemistry ; Blood Proteins ; chemistry ; Cattle ; Cytokines ; biosynthesis ; HEK293 Cells ; Humans ; Interleukins ; biosynthesis ; Lipopolysaccharides ; chemistry ; NF-kappa B ; biosynthesis ; Transfection ; Tumor Necrosis Factor-alpha ; biosynthesis

To explore the anti-tumor proliferation activity of human interleukin-29 (hIL-29) variant and based on bioinformatics analyzed data of hIL-29, a mutant gene hIL-29(mut33,35) was amplified by site-directed mutagenesis and megaprimer PCR. The hIL-29(mut33,35) was inserted into an eukaryotic expression plasmid pPIC9K and successfully expressed in Pichia pastoris GS115. A recombinant variant protein (rhIL-29(mut33,35)) was purified from the ferment supernatant of the engineering GS115. To observe the antineoplastic activity of the variant rhIL-29(mut33,35), a CCK-8 reagent was used to detect the anti-proliferation effect. Results show that it has strong anti-proliferation effect when acted on liver cancer cell BEL7402, colon cancer cell HCT8 and gastric cancer cell SGC7901. The inhibition ratios of the three tumor cells were (30.99 ± 1.58)%, (22.47 ± 1.37)% and (32.05 ± 2.02)%, respectively. In high dose group, the anti-proliferation effect of the rhIL-29(mut33,35) was stronger than that of wild type rhIL-29 (P < 0.01). This indicates the variant rhIL-29(mut33,35) has potential development value for medicine.
Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; drug effects ; Humans ; Interleukins ; biosynthesis ; pharmacology ; Liver Neoplasms ; pathology ; Mutagenesis, Site-Directed ; Pichia ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; pharmacology

To study the chemosensitivity and the mechanisms of recombinant adenovirus vector expressing ING4 and IL-24 (Ad-ING4-IL-24) on lung adenocarcinoma in vitro and in vivo, the expression of ING4 and IL-24 in A549 cells was detected by RT-PCR and Western blotting. The growth inhibition, apoptosis rate and apoptosis body were measured by MTT, flow cytometry and Hoechst staining respectively. For in vivo study, we first established the A549 tumor model by grafting A549 cells in athymic nude mice; and then injected Ad-ING4-IL-24 into the tumors. Two weeks after injection, we killed the mice, removed the tumors, weighted and calculated the ratios of tumor-suppression. We also detected the expressions of ING4, IL-24, bax, bcl-2, VEGF with immunohistochemistry. The results indicated that ING4 and IL-24 were proved successfully transcription and expression in A549 cells. More interestingly, the joint group inhibited the growth of A549 cells and induced apoptosis. The in vivo data showed that the joint group suppressed the tumor growth conspicuously through up-regulating the expression of bax, and down-regulating the expression of bcl-2, VEGF. The study proved that Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug DDP in lung adenocarcinoma, which may related with cell apoptosis and antiangiogenesis.
Adenocarcinoma ; drug therapy ; metabolism ; Adenoviridae ; genetics ; metabolism ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Lung Neoplasms ; drug therapy ; metabolism ; Mice ; Mice, Nude ; Neoplasms, Experimental ; drug therapy ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics

In order to get a better understanding of the role of protease-activated receptor 2 (PAR2) in type 2 helper T (Th2) cell responses against Trichinella spiralis infection, we analyzed Th2 responses in T. spiralis-infected PAR2 knockout (KO) mice. The levels of the Th2 cell-secreted cytokines, IL-4, IL-5, and IL-13 were markedly reduced in the PAR2 KO mice as compared to the wild type mice following infection with T. spiralis. The serum levels of parasite-specific IgE increased significantly in the wild type mice as the result of T. spiralis infection, but this level was not significantly increased in PAR2 KO mice. The expression level of thymic stromal lymphopoietin, IL-25, and eotaxin gene (the genes were recently known as Th2 response initiators) of mouse intestinal epithelial cells were increased as the result of treatment with T. spiralis excretory-secretory proteins. However, the expression of these chemokine genes was inhibited by protease inhibitor treatments. In conclusion, PAR2 might involve in Th2 responses against T. spiralis infection.
Animals ; Antibodies, Helminth/blood ; Chemokine CCL11/biosynthesis ; Cytokines/biosynthesis ; Female ; Gene Expression Profiling ; Immunoglobulin E/blood ; Interleukin-13/secretion ; Interleukin-4/secretion ; Interleukin-5/secretion ; Interleukins/biosynthesis ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Receptor, PAR-2/*metabolism ; Th2 Cells/*immunology ; Trichinella spiralis/*immunology ; Trichinellosis/*immunology

The epithelial growth factor receptor interference (EGFRi) was obtained by synthetic primers. Overlapping PCR was used to produce EGFRi-IL-24 fusion gene, which is linked by Gly4Ser3. After sequence analysis, EGFRi-IL-24 was cloned into expression vector pPIC9k; EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115. Subsequently, positive clone was selected by G418 and PCR, and its phenotype was determined by SDS-PAGE and MTT assay. The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.
Antineoplastic Agents ; pharmacology ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo obtain streptavidin-tagged human interleukin-21 (hIL21) fusion protein and evaluate its bioactivities.

METHODShIL21-SA-pET21 and pET24a-SA- hIL21 plasmids were constructed and expressed in BL21(DE3) host bacteria. The hIL21-SA and SA- hIL21 fusion protein were purified through Ni-NTA affinity chromatography and refolded by dialysis. Flow cytometry was used to detect hIL21-SA and SA- hIL21 fusion protein on the biotinylated MB49 tumor cells. MTT assay was used to evaluate the effect of the fusion protein on the proliferation of human peripheral blood lymphocytes (PBLs) stimulated by Anti-CD3.

RESULTSThe recombinant fusion proteins were highly expressed in BL21(DE3) at about 30% of the total bacterial proteins. The two fusion proteins exhibited bifunctional activities, i.e. both biotin-binding property and hIL21 activity and SA-mediated high-affinity binding to biotinylated cell surfaces (with anchoring modified rate of about 95.18% and 96.91%).

CONCLUSIONWe have successfully obtained bifunctional fusion protein hIL21-SA and SA- hIL21,which will provide a basis for further study of tumor biotherapy using the proteins.

Cancer Vaccines ; immunology ; Cell Line, Tumor ; Escherichia coli ; genetics ; metabolism ; Humans ; Interleukins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Streptavidin ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of respiratory syncytial virus (RSV) infection on the production of thymic stromal lymphopoietin (TSLP) and Th1/Th2 balance in asthmatic mice.

METHODSThirty-two female BALB/c mice were randomly divided into 4 groups, namely the PBS group, ovalbumin (OVA) group, RSV group and OVA/RSV group. The mice were sensitized by OVA and then stimulated with nebulized OVA, and RSV was inoculated into the nasal cavity of the mice. BUXCO noninvasive lung function detection was performed to examine the airway response to metacholine, and enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of IL-4, IL-5, IL-13, and IFN-gamma in the mice. The cells in the bronchoalveolar lavage fluid (BALF) were counted and classified, and the supernatants of the BALF were used for the detection of TSLP. Histopathological changes in the lung tissues of the mice were examined using HE staining, and immunohistochemistry using anti-mouse TSLP antibody was performed to examine TSLP expressions in the airway epithelial cells.

RESULTSRSV infection promoted the production of TSLP in the asthmatic mice, and the concentration of TSLP in OVA/RSV group (2.13-/+0.05 ng/ml) was significantly higher than that in the other groups (P<0.01). RSV infection increased the serum levels of IL-4, IL-5, IL-13, and IFN-gamma in the mice. The total BALF cells, eosinophils, lymphocytes and neutrophils in OVA/RSV group were significantly higher than those in the other groups; noninvasive lung function examination showed higher Penh value in OVA/RSV group (318.66-/+50.87) than in the other groups when the inhaled metacholine increased to 6.25 mg/ml (P<0.01). More obvious and extensive airway inflammatory cell infiltration in OVA/RSV group were observed, and immunohistochemical staining also showed higher expression of TSLP in the airway epithelial cells of OVA/RSV group.

CONCLUSIONSRSV infection promotes the production of TSLP in the airway epithelial cells and increases the level of Th2 cytokines in asthmatic mice. Concurrent RSV infection can exacerbate Th2 inflammatory reaction in asthmatic mice.

Animals ; Bronchoalveolar Lavage Fluid ; Cytokines ; biosynthesis ; secretion ; Female ; Immunohistochemistry ; Inflammation ; immunology ; virology ; Interferon-gamma ; blood ; Interleukins ; blood ; Lung ; immunology ; metabolism ; virology ; Mice ; Mice, Inbred BALB C ; Respiratory Syncytial Virus Infections ; blood ; immunology ; metabolism ; Th2 Cells ; cytology ; immunology ; virology

We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Fusion ; genetics ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Interleukins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Adenoviridae ; genetics ; Cell Death ; Genetic Vectors ; HT29 Cells ; Humans ; Interleukins ; biosynthesis ; genetics ; RNA, Catalytic ; biosynthesis ; genetics ; RNA, Messenger ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism