A Novel Gene of β Chain of the IFN-γ Receptor ofHuiyangChicken: Cloning, Distribution, and CD Assay
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The beta chain of the interferon-gamma receptor (IFNGR-2) plays a critical role in signal transmission to the nucleus by IFN-gamma. Here, we cloned the full-length cDNA of IFNGR-2 of Huiyang chicken using RACE. mRNA transcripts of IFNGR-2 were detected in peripheral blood leukocytes (PBL) and various organs using Northern blot analysis. The extracellular region of IFNGR-2 (IFNGR-2EC) was expressed in Pichia pastoris, and its secondary structure was investigated by circular dichroism (CD). The Huiyang chicken IFNGR-2 gene is 2221 bp with a polyA+ tail, and it encodes 334 amino acids sharing 30%-33% identity with that of rat, mouse, and human IFNGR-2. IFNGR-2 is localized on chromosome 1 of chicken in tandem with IFNAR-1, interleukin- 10 receptor (IL-10R-2), and IFNAR-2. IFNGR-2 was highly expressed in PBL, muscle, spleen, thymus, and cecal tonsil, whereas its expression in cardiac muscle, cloacal bursa, liver, and kidney was comparatively low. Recombinant protein of IFNGR-2EC expressed in P. pastoris formed the secondary structure including 19.8% alpha-helix, 29.6% beta-sheet, 19.7% turn, and 30.9% random. The data show that Huiyang chicken IFNGR-2 shares properties of the IFN receptor family in gene structure and distribution in multiple tissues and PBL. CD analysis indicated that the recombinant protein of IFNGR-2EC resembles the known structure of human IFN receptors.Aim To achieve high-level expression of recombinant hCTRP2 in Pichia pastoris via high-density fermentation.Methods Recombinant hCTRP2 was expressed in Pichia pastoris by high-density fermentation,purified by Ni-NTA affinity chromatography and the bioactivities of purified rhCTRP2 were assayed in vitro test and in vivo test.Results Recombinant transformants with high-level expression of rhCTRP2 were identified and 560 mg·L-1total protein was secreted in 14-L fermentor induction with methanol.The recombinant hCTRP2 proteins were purified using NiNTA affinity chromatography with a yield of about 10.4 mg purified protein from 100 ml culture supernatants.The purified recombinant was assayed to be active by in vitro test and in vivo test.Conclusion The active recombinant hCTRP2 is high-level expressed by fedbatch fermentation and purified with Ni-NTA affinity chromatography.
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The hLYZ (human lysozyme) gene was chemically synthesized, then constructed into yeast secretion expression vector pPICZα. The constructed plasmid was linearized by Sac I digestion and transformed into Pichia pastoris strain GS115 by electroporation method. Nine recombinant strains were successfully screened out by Zeocin and identified by PCR analysis. After induction by methanol, the supernatant from the induced recombinant P. pastoris strain was collected, then analyzed by SDS-PAGE and Western blot analyses. The results showed that the recombinant hLYZ protein was successfully expressed and the expression level reached the peak at 60 h after induction. The recombinant hLYZ was purified by Ni+ affinity chromatography and its yield was 324 mg·L-1 by Bradford assay. The bioactivity of the recombinant hLYZ was determined to be 4473 U·mL-1 by turbidimetric method. The successful expression of the bioactive recombinant hLYZ using P. pastoris expression system and the exploration of the simple and efficient purification and bioactivity measurement method of the recombinant protein provide the basis for large-scale production of recombinant hLYZ using P. pastoris system.
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Aim Expression and analysis of recombinant chicken IL-18 in Pichia pastoris. Methods Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR, and was subcloned into Pichia pastoris expression vector pPICZalphaA to construct the recombinant plasmid pPICZalphaA-ChIL-18. After identified by restriction enzymes digestion analysis, PCR and DNA sequencing, the recombinant plasmid was transformed into Pichia pastoris X-33.Then choosing the multi-copy recombinant strains to be induced for expression.Then the bioactivity of rchIL-18 was analysed by Western blot, ELISA and MTT after purified by Sephadex G-100 column. Results The chicken IL-18 with the immunogenicity was secreted by Pichia pastoris. It could induce T lymphocytes proliferation and secreting IFN-gamma in vitro. Conclusion The chicken IL-18 with obvious biological activity is secreted by Pichia pastoris X-33.
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Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR,and subcloned into Pichia pastoris expression vector pPICZαA,thus constructing the recombinant plasmid pPICZαA-ChIL-18.After identified by restriction enzymes digestion,PCR and DNA sequencing,the recombinant plasmid was transformed into P.pastoris X-33.Then the multi-copy recombinant strains were induced for expression.The expression conditions were further optimized by analyzing the influences of pH value,inducer concentration,inducing time,and inducing temperature on the yields of target protein.The results showed that chicken IL-18 could be secreted by the recombinant P.pastoris and the expression level of recombinant protein was highest under pH 6.0,1.5% of methanol,temperature 26 ℃,and 96 hours of inducing time.This study laid a foundation for the large-scale fermentation of chicken IL-18.
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Objective To construct the recombinant procarboxypeptidase B in Pichia pastoris.Methods The yeast plasmid containing procarboxypeptidase B coding gene was constructed,and then transferred to Pichia pastoris.The positive clone was obtained by G418 resistance selection in His-medium.The rabbit anti-rat recombinant procarboxypeptidase B serum was prepared with recombinant procarboxypeptidase B expressed in E.coli.Western-blotting analysis was used to determine the expressed protein with the prepared antiserum after fermentation and methanol induction of recombinant yeast.Results The rabbit anti-rat recombinant procarboxypeptidase B serum with higher antibody titer was got.The result of Western-blotting analysis showed that the supernatant was positive with the antiserum after methanol induction of recombinant yeast.Conclusion The recombinant procarboxypeptidase B in Pichia pastoris can be constructed with the successfulsecretion of recombinant procarboxypeptidase B.
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Heterologous expression
Heterologous
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Molecular mass
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To study the characteristic of recombinant Cysticerus cellulosae antigen cC1 in Pichia pastoris.The recombinant cC1 was analyzed by SDS-PAGE, FPLC-DEAE Sepharose FF,FPLC-Superdex75,IEF,dephosphorization,sequencing of amino-N-terminal and immunization to mice.MW of the recombinant cC1 was larger than that of recombinant cC1 in E coli.The cC1 protein was easy to purify.The pI was lower than the theorial pI value of cC1 because of phosphorization.The amino-N-terminal carried 4 aminoes from the expression plasmid.Mice test showed r-cC1 had higher immunogenicity in Pichia pastoris than in E coli.Characteristic of recombinant Cysticerus cellulosae antigen cC1 in Pichia pastoris varies from that of recombinant cC1 in E coli.The easy-purification and high- immunogenicity suggests that Pichia pastoris expression system be quite adapt to produce recombinant sub-unit vaccine.
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To identify antiviral activity and the secondary structure of the recombinant protein of Huiyang chicken IFN-γ (chIFN-γ2) expressed in Pichia pastoris,the cDNA encoding mature peptide of IFN-γ was subcloned from plasmid pGEM-T-chIFN-γ2 and recombined with vector pPICZαB.The recombinant plasmid confirmed by enzyme digestion,PCR amplification and sequencing was transformed into P.pastoris GS115.After methanol induction,SDS-PAGE analysis of the culture supernatant of recombinant yeast strains indicated that the yield of recombinant IFN-γ was 0.604mg/mL and the molecular weight was(approximately) 33ku.The recombinant IFN-γ expressed in P.pastoris had antiviral activity of 3.2×10~4(U/mg) in CEF-VSV.The secondary structure of the recombinant IFN-γ was investigated by Circular Dichroism,and contents of α helix,β sheet,turn,and random were 55.1%,8.3%,13.9% and 22.8%,respectively.These results showed that the recombinant IFN-γ with high antiviral activity was expressed successfully in P.pastoris and the chicken IFN-γ was a helical protein which was similar to IFN-γ of mammals.
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Aim:To express the recombinant peptide from gp52 and pp150 C-terminal peptides of human cytomegalovirus (HCMV) in the Pichia pastoris GS115. Methods: After linearised by Sac Ⅰ or Bgl Ⅱ, respectively, the recombinant plasmids were transformed into Pichia pastoris GS115 by electroporation. The Mut+ and Muts transformants were screened according to their different growth characteristic in MD plates without histidine. Some of positive transformants were confirmed by PCR. All Mut+ and Muts clones were induced with methanol. After 4 days of methanol induction, the dialyzed and lyophilizated products were analyzed by SDS-PAGE and Western blot. Results: Recombinant peptied of HCMV was expressed effectively in Mut+ Pichia pastoris and camp up to 76.5% of total proteins in supernatant. Conclusion: High-level expression of secreted recombinant peptide of HCMV has been successfully archived in Pichia pastoris expression system.
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Objective:To construct a recombinant plasmid PHIL-D2/K1-3 for the expression of human AngiostatinK1-3 in Pichia pastoris.Methods :The gene of human AngiostatinK1-3 was amplified from the embryo liver tissue using RT-PCR and then cloned into PHIL-D2 vector.After being linearized,the recombinant plasmid was transformed into Pichia pastoris GS115.The positive transformant was identified by PCR and Southern blotting and then induced by methanol.The expressed products was analyzed by SDS-PAGE(12%) and Western blot.After being purified by lysine/Sepharose-4B affinity chromatography,the recombinant protein concentration was determined with Lowry's method and its antiangiogenic activity was assayed as well.Results: The recombinant protein was about 30 KD.and the level of expression was 6.8mg/L.In the activity assay,the recombinant protein could strongly inhibit the proliferation of HUVEC.Conclution: The recombinant human AngiostatinK1-3 is successfully expressed in Pichia pastoris GS115.
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Angiostatin
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