Expression of bovine interferon-γ in Escherichia coli and Pichia pastoris and comparison of their antiviral activities

2006 ◽  
Vol 3 (1) ◽  
pp. 39-46
Author(s):  
Shi Xi-Ju ◽  
Zhang Can ◽  
Han Chun-Lai ◽  
Xia Chun ◽  
Wang Ming
Keyword(s):  
Escherichia Coli ◽  
Pichia Pastoris ◽  
Cell Line ◽  
Chicken Embryo Fibroblast ◽  
Interferon Γ ◽  
Peripheral Blood Lymphocytes ◽  
Methanol Induction ◽  
E Coli ◽  
Antiviral Activities ◽  
Vesicular Stomatitis

AbstractThe bovine interferon-γ gene cloned from total RNA of peripheral blood lymphocytes stimulated with concanavalin A (ConA), using the reverse transcriptase-polymerase chain reaction (RT-PCR), was cloned into pGEM T-easy vector and sequenced. The mature peptide without signal peptide was then subcloned and constructed into the expression plasmids pET28a/BoIFN-γ and pPICZα/BoIFN-γ, respectively. The former was highly expressed, induced by isopropyl β-d-thiogalactoside (IPTG), in Escherichia coli BL21, with 30% of bacterial proteins in inclusion bodies. The latter was expressed in Pichia pastoris GS115 with methanol induction. The expressed products were secreted directly into cultural supernatant at concentrations of 1.0 g/l. The same recombinant proteins had antiviral activities 10 times higher in an MDBK (Madan-Darby bovine kidney)/VSV (Vesicular stomatitis virus) cell line than in a CEF (chicken embryo fibroblast)/VSV) cell line, and the antiviral activities expressed in P. pastoris were higher than those expressed in E. coli in the same cell line.

scholarly journals P fimbriae and other adhesins enhance intestinal persistence of Escherichia coli in early infancy

1998 ◽  
Vol 121 (3) ◽  
pp. 599-608 ◽  
Author(s):  
I. ADLERBERTH ◽  
C. SVANBORG ◽  
B. CARLSSON ◽  
L. MELLANDER ◽  
L.-Å. HANSON ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Line ◽  
Intestinal Epithelial Cells ◽  
Antigen Expression ◽  
Intestinal Epithelial ◽  
E Coli ◽  
O Antigen ◽  
Ht 29 ◽  
Intestinal Persistence

Resident and transient Escherichia coli strains were identified in the rectal flora of 22 Pakistani infants followed from birth to 6 months of age. All strains were tested for O-antigen expression, adhesin specificity (P fimbriae, other mannose-resistant adhesins or type 1 fimbriae) and adherence to the colonic cell line HT-29. Resident strains displayed higher mannose- resistant adherence to HT-29 cells, and expressed P fimbriae (P=0·0036) as well as other mannose-resistant adhesins (P=0·012) more often than transient strains. In strains acquired during the first month of life, P fimbriae were 12 times more frequent in resident than in transient strains (P=0·0006). The O-antigen distribution did not differ between resident and transient strains, and none of the resident P-fimbriated strains belonged to previously recognized uropathogenic clones. The results suggest that adhesins mediating adherence to intestinal epithelial cells, especially P fimbriae, enhance the persistence of E. coli in the large intestine of infants.

CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5202-5211 ◽  
Author(s):  
Karen E. Anderson ◽  
Keith B. Boyle ◽  
Keith Davidson ◽  
Tamara A. M. Chessa ◽  
Suhasini Kulkarni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Nadph Oxidase ◽  
Cell Line ◽  
Microbial Pathogens ◽  
Class I ◽  
Human Neutrophils ◽  
Class Iii ◽  
Single Class ◽  
E Coli

Abstract Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the β2-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns(3)P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3)P binding to the PX domain of their p40phox oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns(3)P-regulation of the oxidase must exist.

scholarly journals The Effect of Interferon-γ and Lipopolysaccharide on the Growth of Francisella tularensis LVS in Murine Macrophage-like Cell Line J774

2009 ◽  
Vol 52 (3) ◽  
pp. 101-106 ◽  
Author(s):  
Monika Holická ◽  
Jakub Novosad ◽  
Martina Loudová ◽  
Manuela Kudlová ◽  
Jan Krejsek
Keyword(s):  
Cell Line ◽  
Francisella Tularensis ◽  
Interferon Γ ◽  
Growth Potential ◽  
Protective Effects ◽  
Recombinant Interferon ◽  
E Coli ◽  
J774 Cells ◽  
J774 Cell Line ◽  
Stimulation Of

Background: Francisella tularensis, a causative agent of human tularemia, displaying the ability to proliferate inside the human cells. Aims: To evaluate the growth potential of F. tularensis LVS strain in macrophage-like cell line J774 modulated by recombinant interferon γ and E. coli derived lipopolysaccharide. Results: Stimulation of J774 cells either by interferon-γ or lipopolysaccharide alone, or especially in combination before infection F. tularensis, revealed protective effects. Higher concentrations of stimulating agents were needed to inhibit ongoing F. tularensis infection. Conclusions: Stimulation of J774 cell line by combination of interferon-γ with lipopolysaccharide inhibits the intracellular growth of F. tularensis.

scholarly journals Ekspresi Prethrombin-2 Manusia Recombinan dalamPichia pastoris dan Optimasi Kondisi Ekspresinya

2017 ◽  
Vol 4 (1) ◽  
pp. 27
Author(s):  
Shabarni Gaffar ◽  
Purba Upay ◽  
Iman Permana Maksum ◽  
Khomaini Hasan ◽  
Toto Subroto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pichia Pastoris ◽  
E Coli

Pretrombin merupakan prekursor dari trombin yang memiliki aktivitas proteolitik. Trombin merubah fibrinogen menjadi benang fibrin yang salah satu aplikasinya adalah dapat digunakan sebagai lem untuk menggantikan teknik jahitan pasca bedah. Aplikasi trombin untuk pembuatan lem fibrin menuntut diproduksinya trombin rekombinan. Tujuan dari penelitian ini adalah ekspresi gen pretrombin-2 (PT2) manusia rekombinan menggunakan sistem ekspresi Pichia pastoris. Gen pengode PT2 dirancang sesuaidengan kodon preferensi P. pastoris. Fragmen PT2 diamplifikasi dengan metoda PCR dengan penambahan sisi restriksi EcoR1 pada ujung 5’ dan sisi restriksi SacII pada ujung 3’. Produk PCR yang berukuran 924 pb diligasi dengan vektor ekspresi pPICZaB untuk P. pastoris dan disubkloning dalam inang Escherichia coli. Urutan nukleotida dikonfirmasi dengan metoda dideoxy Sanger. Plasmid rekombinan pPICZaB-PT2 kemudian digunakan untuk mentransformasi P. pastoris SMD1168 defisien protease dengan metoda elektroporasi. Hasil penelitian menunjukkan bahwa gen PT2 berhasil diamplifikasi dan dikloning dalam E. coli. Analisis restriksi dan penentuan urutan DNA menunjukkan bahwa PT2 rekombinan 100% homologi dengan hasil rancangan. Hasil ekspresi PT2 oleh P. pastoris menggunakan metanol sebagai inducer memperlihatkan bahwa PT2 dengan berat molekul 35 kDa berhasil diekspresikan. Optimasi kondisi ekspresi melalui variasi konsentrasi metanol sebagai inducer dan sorbitol sebagai sumber karbon tambahan menunjukkan bahwa metanol 2% dan sorbitol 2% merupakan kondisi optimum ekspresi PT2.

scholarly journals Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9010
Author(s):  
Yonggang Wang ◽  
Jiangqin Wang ◽  
Feifan Leng ◽  
Jianzhong Ma ◽  
Alnoor Bagadi
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Glucose Oxidase ◽  
Fermentation Medium ◽  
Logarithmic Phase ◽  
Transgenic Strain ◽  
E Coli ◽  
Cell Lysate

The gene encoding glucose oxidase from Aspergillus niger ZM-8 was cloned and transferred to Pichia pastoris GS115, a transgenic strain P. pastoris GS115-His-GOD constructed. The growth curve of P. pastoris GS115-His-GOD was consistent with that of Pichia pastoris GS115-pPIC9K under non-induced culture conditions. Under methanol induction conditions, the growth of the GOD-transgenic strain was significantly lowered than P. pastoris GS115-pPIC9K with the induced-culture time increase, and the optical densities of GOD-transgenic strain reached one-third of that of the P. pastoris GS115-pPIC9K at 51 h. The activity of glucose oxidase in the cell-free supernatant, the supernatant of cell lysate, and the precipitation of cell lysate was 14.3 U/mL, 18.2 U/mL and 0.48 U/mL, respectively. The specific activity of glucose oxidase was 8.3 U/mg, 6.52 U/mg and 0.73 U/mg, respectively. The concentration of hydrogen peroxide formed by glucose oxidase from supernatant of the fermentation medium, the supernatant of the cell lysate, and the precipitation of cell lysate catalyzing 0.2 M glucose was 14.3 μg/mL, 18.2 μg/mL, 0.48 μg/mL, respectively. The combination of different concentrations of glucose oxidase and glucose could significantly inhibit the growth of Agrobacterium and Escherichia coli in logarithmic phase. The filter article containing supernatant of the fermentation medium, supernatant of the cell lysate, and precipitation of cell lysate had no inhibitory effect on Agrobacterium and E. coli. The minimum inhibitory concentration of hydrogen peroxide on the plate culture of Agrobacterium and E. coli was 5.6 × 103 μg/mL and 6.0 × 103 μg/mL, respectively.

scholarly journals Comparative Analysis of Attachment of Shiga-Toxigenic Escherichia coli and Salmonella Strains to Cultured HT-29 and Caco-2 Cell Lines

2009 ◽  
Vol 75 (6) ◽  
pp. 1796-1799 ◽  
Author(s):  
Glen E. Mellor ◽  
Rebecca M. Goulter ◽  
T. W. Raymond Chia ◽  
Gary A. Dykes
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Monolayers ◽  
E Coli ◽  
Ht 29

ABSTRACT The ability of Escherichia coli and Salmonella isolates to attach to Caco-2 and HT-29 cell monolayers was measured. All isolates displayed a greater ability to attach to Caco-2 cells than HT-29 cells, and overall E. coli isolates attached better to both cell lines than Salmonella isolates. Bacteria that were considered to be pathogenic displayed no greater ability to attach to cell lines than those that were not considered to be pathogenic. Additionally, no correlation was found between cell line attachment and previously determined hydrophobicity results.

scholarly journals METODOLOGIA PARA EXPRESSÃO DA GLICOPROTEÍNA S1 DO VÍRUS DA BRONQUITE INFECCIOSA DAS GALINHAS EM SISTEMAS PROCARIOTO E EUCARIOTO

2016 ◽  
Vol 4 (1) ◽  
pp. 81
Author(s):  
Paula Fonseca Finger ◽  
Luana Alves Dummer ◽  
Michele Soares Pepe ◽  
Carolina Georg Magalhães ◽  
Paulo Augusto Esteves ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pichia Pastoris ◽  
Western Blotting ◽  
Rt Pcr ◽  
E Coli ◽  
Dot Blotting

A bronquite infecciosa das galinhas (IB) é uma enfermidade altamente contagiosa que acomete aves de todas as idades e causa grandes prejuízos na avicultura. O trabalho descreve uma metodologia para expressão da glicoproteína S1 do vírus da bronquite infecciosa das galinhas (IBV) em Pichia pastoris e Escherichia coli. Para P. pastoris o gene foi amplificado por RT-PCR, com posterior clonagem e expressão no vetor pPICZαB. Para E. coli, foi elaborado o gene sintético a partir de uma sequência consenso de amostras de campo nacionais e internacionais, clonado e expresso no vetor pAE. Após indução dos clones, a proteína S1 foi identificada pelas técnicas de Dot blotting e Western blotting. A proteína expressa apresentou reatividade frente a soros de aves positivas para IBV, evidenciando sua antigenicidade.

scholarly journals Preliminary Characterization of the Transcriptional Response of the Porcine Intestinal Cell Line IPEC-J2 to EnterotoxigenicEscherichia coli,Escherichia coli, andE. coliLipopolysaccharide

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Marisa M. Geens ◽  
Theo A. Niewold
Keyword(s):  
Escherichia Coli ◽  
Cell Line ◽  
Transcriptional Response ◽  
Intestinal Cell ◽  
Adhesion Assay ◽  
Transcriptional Level ◽  
Suitable Model ◽  
E Coli ◽  
Intestinal Cell Line

IPEC-J2, a promisingin vitromodel system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenicEscherichia coli(ETEC), nonpathogenicE. coli, andE. coliendotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured withE. colistrains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction.

scholarly journals The Adherent/Invasive Escherichia coli Strain LF82 Invades and Persists in Human Prostate Cell Line RWPE-1, Activating a Strong Inflammatory Response

2016 ◽  
Vol 84 (11) ◽  
pp. 3105-3113 ◽  
Author(s):  
Maria P. Conte ◽  
Marta Aleandri ◽  
Massimiliano Marazzato ◽  
Antonietta L. Conte ◽  
Cecilia Ambrosi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inflammatory Response ◽  
Host Cell ◽  
Cell Line ◽  
Neonatal Meningitis ◽  
Prostate Cell ◽  
Content Type ◽  
E Coli ◽  
Prostate Cell Line ◽  
Strong Inflammatory Response

Adherent/invasiveEscherichia coli(AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenicE. coli(ExPEC), neonatal meningitis-associatedE. coli(NMEC), and uropathogenicE. coli(UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers.In silicoanalysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Ourin vitrodata support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract.

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