Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.
Electroporation ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; analysis ; biosynthesis ; genetics

OBJECTIVETo research the effects of recombinant human beta-defensin-3 (hBD-3) on expression of interleukin-17A (IL-17A) and interleukin-22 (IL-22) in BEAS-2B cell.

METHODSThe BEAS-2B cells were stimulated with different concentrations of hBD-3 for 6 hours and 24 hours, respectively. Toll-like receptor 2 (TLR2), IL-17A and IL-22 mRNA expression levels were determined by real-time PCR, and the expression levels of IL-17A and IL-22 protein were examined by enzyme linked immune-sorbent assay.

RESULTSTLR2 mRNA in BEAS-2B cells were significantly increased in a concentration-and time-dependent manner after stimulating by hBD-3 for 24 hours compared to 6 hours. The IL-17A has significantly increased in mRNA and protein levels stimulated 24 hours in a concentration of 100 ng/ml, however, IL-17A mRNA expression has increased while protein didn't change stimulated 6 hours in a concentration of 50 ng/ml. The IL-22 mRNA and protein expression reached peak levels after stimulating in a concentration of 50 ng/ml of hBD-3 while IL-22 expression declined in mRNA and protein levels as the concentration of hBD-3 increased.

CONCLUSIONSRecombinant hBD-3 can up-regulated the expression of TLR2, IL-17A and IL-22, lower concentration of hBD-3 mainly increased the expression of IL-22 while higher concentration of hBD-3 mainly increased the expression of IL-17A. These results show that different concentrations of hBD-3 maybe activate different transcription factors which was mediated by TLR2, initiating host immune response.

Cell Line ; Humans ; Interleukin-17 ; genetics ; metabolism ; Interleukins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Toll-Like Receptor 2 ; genetics ; metabolism ; beta-Defensins ; genetics ; metabolism

To clone human interleukin-26 (hIL-26) and express it in E. coli efficiently. Two pairs of primers were synthesized according to the hIL-26 gene reported on GenBank. The hIL-26 gene was cloned by nest PCR following the first round RT-PCR from human peripherial blood monocytes total RNA, and then the PCR product was cloned into pMD18-T vector. Colony PCR, restriction analysis and sequence analysis showed that the gene cloned was the same as the reported hIL-26. The recombinant was cut with BamHI and EcoR I to obtain the hIL-26 fragment, and then the fragment was inserted into pBV220 which was cut with the same enzymes. The recombinant expression vector was induced to express hIL-26 at 42 degrees C, SDS-PAGE analysis showed that the recombinant protein accounted for up to 20% of the whole protein of E. coli, and the protein was also confirmed by Western blotting. Purity of the protein was found to be above 90% after purified with molecular sieve. After renaturalized with glutathione buffer, the promoting effect of it on the production of IFN-y in PBMC was detected by RT-PCR. A recombinant bacterial strain for expressing hIL-26 with biological activity was constructed successfully.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Interleukins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

The melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) is a novel candidate of tumor suppressor, which can experimentally inhibit the proliferation of cancer cells and induce apoptosis of various human malignant cells.
Apoptosis ; Cell Proliferation ; Genetic Therapy ; methods ; Humans ; Interleukins ; biosynthesis ; genetics ; Neoplasms ; metabolism ; pathology ; therapy ; Tumor Suppressor Proteins ; biosynthesis ; genetics

To investigate the inhibitory effect and anti-cancer mechanism of adenovirus mediated IL-24 gene expression on the human U251 glioma cell. U251 glioma cells were infected with Ad-IL-24 at various multiplicity of infection (MOIs). Cell proliferation was determined by MTT assay. Cell apoptosis was detected by flow cytometry and Hochest staining. The transcription of apoptosis-related genes was analyzed by reverse transcription-PCR (RT-PCR), and the expression of Cleaved Caspase-3 was analyzed by Western blotting. The result showed that the growth of U251 glioma cells was significantly inhibited by Ad-IL-24 at the MOI of 100. The apoptotic rate of U251 glioma cells was 42% 72 h after infection with Ad-IL-24. Four days after infection, the growth of the U251 glioma cells was inhibited to 50%. RT-PCR showed that Ad-IL-24 not only up-regulated expression of bax/bcl-2, ICE, C-myc, p53 and down-regulated the expression of HIF-1alpha, but also enhanced Caspase-3 activation, eventually resulting apoptosis. Taken together, these results suggest that infection of U251 glioma cells with Ad-IL-24 can inhibit growth and induce apoptosis significantly by the regulation of apoptosis-related genes.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; Brain Neoplasms ; genetics ; pathology ; Cell Proliferation ; drug effects ; Genetic Therapy ; Glioma ; genetics ; pathology ; Humans ; Interleukins ; genetics ; metabolism ; Recombination, Genetic ; Tumor Cells, Cultured

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

OBJECTIVE: To investigate the expression of EBI3 and p28 mRNA (the 2 subunits of IL-27) in the brain and spinal cord of the model of experimental autoimmune encephalomyelitis (EAE), and to explore their effect on EAE. METHODS: Seventy-two adult female SPF C57BL/6J mice (inbred strain) were randomly divided into a control group, an adjuvant group, and an EAE group. RT-PCR was performed to detect the expression of EBI3 mRNA and p28 mRNA in the brain and spinal cord. RESULTS: The expression of EBI3 mRNA and p28 mRNA was up-regulated at onset in the EAE group, which increased quickly during peak phase and maintained at a high level in the chronic phase. There was significant difference in the expression of EBI3 and p28 mRNA between the EAE group and the control/adjuvant group (P<0.01). Additionally, there was no remarkable difference in the expression of EBI3 and p28 mRNA in the brain and spinal cord between the control group and the adjuvant group (P>0.05). CONCLUSION IL-27 may play a role of promoting the morbility of EAE in the early stage, and sustain the inflammatory response in endgame.
Animals ; Brain ; metabolism ; Chronic Disease ; Encephalomyelitis, Autoimmune, Experimental ; metabolism ; Interleukins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Minor Histocompatibility Antigens ; Protein Subunits ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, Cytokine ; genetics ; metabolism ; Spinal Cord ; metabolism

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

OBJECTIVES: To investigate the expression of interleukin-37 (IL-37), vascular endothelial growth factor A (VEGFA), and transforming growth factor-β1 (TGF-β1) in children with primary immune thrombocytopenia (ITP) and their correlation with T cells. METHODS: A retrospective analysis was conducted on 45 children with ITP (ITP group) who were admitted to Handan Central Hospital from January 2020 to April 2022, and 30 healthy children who underwent physical examination during the same period were included as the healthy control group. The mRNA expression levels of IL-37, VEGFA, and TGF-β1 and the levels of regulatory T cells (Treg) and helper T cells 17 (Th17) were measured before and after treatment, and the correlation between the mRNA expression levels of IL-37, VEGFA, and TGF-β1 and the levels of Treg, Th17, and Treg/Th17 ratio were analyzed. RESULTS: Compared with the healthy control group, the ITP group had a significantly higher mRNA expression level of IL-37 and a significantly higher level of Th17 before and after treatment, as well as significantly lower mRNA expression levels of VEGFA and TGF-β1 and significantly lower levels of Treg and Treg/Th17 ratio (P<0.05). After treatment, the ITP group had significant reductions in the mRNA expression level of IL-37 and the level of Th17 and significant increases in the mRNA expression levels of VEGFA and TGF-β1 and the levels of Treg and Treg/Th17 ratio (P<0.05). Correlation analysis showed that in the ITP group, the mRNA expression levels of IL-37 and TGF-β1 were negatively correlated with the levels of Treg and Treg/Th17 ratio (P<0.05) and were positively correlated with the level of Th17 (P<0.05) before and after treatment; the mRNA expression level of VEGFA was positively correlated with the levels of Treg and Treg/Th17 ratio (P<0.05) and was negatively correlated with the Th17 level (P<0.05) before and after treatment. CONCLUSIONS Abnormal expression levels of IL-37, VEGFA, and TGF-β1 may be observed in children with ITP, which is significantly associated with the imbalance of Treg/Th17 ratio. It is speculated that the cytokines such as IL-37, VEGFA, and TGF-β1 may be involved in the development and progression of ITP or may become important potential targets for the treatment of children with ITP. Citation:Chinese Journal of Contemporary Pediatrics, 2023, 25(11): 1131-1136.
Child ; Humans ; Interleukins ; Purpura, Thrombocytopenic, Idiopathic ; Retrospective Studies ; RNA, Messenger/metabolism* ; T-Lymphocytes, Regulatory ; Th17 Cells/metabolism* ; Transforming Growth Factor beta1/genetics* ; Vascular Endothelial Growth Factor A/genetics*

Study effect and mechanisms of growth-suppression of hepatocelluar carcinoma (HCC) in nude mice. The construction of the pAdeasy-1-pTrack-CMV-hIL-24 recombined adenovirus vector (Ad-hIL-24) was completed and lineared with PacI. Ad-hIL-24 were transfected into QBI-293 cells and obtained. 16 nude mice of the subcutaneous tumor models were established with SMMC-7721 HCC and were randomly divided into NS, 5-Fu, Ad and Ad-hIL-24 groups. Then 100 microL NS, Ad (10(7) pfu) and Ad-hIL-24 (10(7) pfu) for each one were given respectively QOD, and 5-Fu (20 microg/kg) were injected Q.D., for 5 times, with intratumor injections. After 15 d, 16 mice were sacrificed and subcutaneous tumors were taken out. The volumes (before administration, 1 week and 2weeks after administration) were measured and the weights of tumor were weighed and ratios of tumor-suppression were calculated. The morphological changes of apoptotic tumor cells were observed under microscope. Caspase3, P53 and P27, CD34 and VEGF were tested in immunohistochemistry. In tumor subcutaneous model, compared with NS group, the ratios of tumor-suppression of Ad-hIL-24 group and 5-Fu group were 68.52% (P < 0.01) and 65.64 (P < 0.01), respectively. Caspase3 protein in Ad-hIL-24 group was higher than other 3 groups significantly (P < 0.01). The expression of P27 also differed from NS group (P < 0.01). CD34 and VEGF protein in Ad-hIL-24 group can inhibit neovascularization obviously (P < 0.001), compared with NS and Ad groups. Ad-hIL-24 inhibits the growth of SMMC-7721 HCC on nude mice's. The mechanisms of tumor-suppression may be multi-pathways such as the induction of caspase3 pathway, P27 activities and the antiangiogenic mechanism.
Adenoviridae ; genetics ; Animals ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; genetics ; Humans ; Immunohistochemistry ; Interleukins ; genetics ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Mice ; Mice, Nude ; Plasmids ; genetics