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Objective: To explore the functions of pathogenesis related protein 1-like (PR1-like) in suspension cell of Sorbus aucuparia (SaPR1-like) under biotic stress. Method: The full sequence of SaPR1-like gene was cloned and analyzed by multiple bioinformatic tools. The expression of SaPR1-like in the suspension cell of S. aucuparia in response to harpin protein stress was analyzed by real-time fluorescence quantitative polymerase chain reaction (PCR). Result: A SaPR1-like gene was identified and cloned, which contained a complete open reading frame and encoded 161 amino acids. The coding protein was hydrophilic and stable, had transmembrane structure and signal peptide, and belonged to secretory protein. Amino acid sequence alignment data indicated that the C-terminal of SaPR1-like contained a highly conserved domain[cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1 peptides (CAPE)], which could induce defense genes to produce immune responses against biotic stresses. SaPR1-like gene expression was significantly increased after harpin protein induced suspension cell of S. aucuparia for 24 h. Conclusion: A SaPR1-like gene derived from the PR1 family is found to induce significant responses to biotic elicitors in S. aucuparia. This study highlights a role for PR1 in immune signaling and suggests the potential application of PR1 in efforts to defeat biotic stress in plants.
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Astragalus membranaceus pathogenesis-related protein 10 (AmPR-10) is largely expressed in case of environmental pressure and pathogen invasion. This study aims to explore the biochemical functions of AmPR-10. The dried root of Astragalus membranaceus was mechanically homogenized and extracted by Tris-HCl buffer to obtain its crude extract, which was then purified by anion exchange chromatography and gel filtration chromatography to obtain electrophoretically pure AmPR-10. The nuclease activity of AmPR-10 was tested with different RNAs by detecting the absorption value at 260 nm. The results demonstrated potent nuclease activity toward yeast tRNA, yeast RNA, Poly (A) and Poly (C). The optimum reaction temperature was 50 °C and pH was 7-8. EDTA showed no effect on its activity, while Mg²⁺ exhibited potent activation effect on the activity, and Co²⁺, Ca²⁺ and Zn²⁺ manifested moderately inhibition of the activity. Since AmPR-10 had no sequence homology with other known nucleases, AmPR-10 was probably a novel nuclease. The inhibition kinetic data against papain was analyzed by Lineweaver-Burk plots, and the results showed that the inhibition of papain followed noncompetitive-type kinetics. AmPR-10 played an important role in Astragalus membranaceus defense mechanism against environmental pressure and pathogen invasion, which may be achieved by inhibiting cycteine enzymes activity.
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Base on the transcriptome analysis and RT-PCR techniques,a pathogenesis-related protein 10 gene was isolated from Panax notoginseng root and named as PnPR10-2. Bioinformatics and phylogenetic trees analysis revealed that open reading frame (ORF) of PnPR10-2 was 465 bp in length,encoding 154 amino acids,containing one typical conserved domain of pathogenesis related protein Bet v I family, and showed high similarity with that from P. ginseng. The recombinant expressed plasmid pET32a(+)-PnPR10-2 was expressed in Escherichia coli BL21. The expression conditions were optimized and it could be expressed well in soluble and inclusion body protein. Purified PnPR10-2 recombinant protein from the supernatant of cells was used to analysis the pathogen resistance activity by paper method. The purified recombinant protein could inhibit typical root rot disease pathogen (Fusarium solani and Cylindrocarpon destructans）growth evidently, we conjecture that PnPR10-2 may participated in defense response of P. notoginseng resistance to root rot disease pathogen.
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Objective To express a recombinant protein TFPR1 ( the functional region of the snake venom proteins from Trimeresurus flavoviridis) in Pichia pastoris expression system. Methods The target gene was codon-optimized and synthesized according to the sequence of the conserved structural do-main of triflin and then cloned into the Pichia pastoris expression vector pPICZαA to construct the recombi-nant expression plasmid pPICZαA-TFPR1. The recombinant plasmid pPICZαA-TFPR1 was electroporated into the yeast strain X33. The transformed strains carrying expression plasmid were screened out with Zeocin and then induced by methanol to express the recombinant protein TFPR1. ELISA was performed for the screening of positive clones. SDS-PAGE and Western blot were used for further identification of the ex-pressed products. Results The recombinant plasmid pPICZαA-TFPR1 was successfully constructed. The recombinant protein TFPR1 was expressed in a secreted form at a molecular weight of 16×103. Conclusion The recombinant protein TFPR1 was successfully expressed in Pichia pastoris expression system, which laid a foundation for further researches on its biological function and application as an adjuvant.
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After glioma pathogenesis-related protein 1 (GLIPR1/Glipr1) was identified, the expression of GLIPR1 was shown to be down-regulated in human prostate cancer, owing in part to methylation in the regulatory region of this gene in prostate cancer cells. Additional studies showed that GLIPR1/Glipr1 expression is induced by DNA-damaging agents independent of p53. Functional analysis of GLIPR1 using in vitro and in vivo gene-transfer approaches revealed both growth suppression and proapoptotic activities for mouse Glipr1 and human GLIPR1 in multiple cancer cell lines. The proapoptotic activities were dependent on production of reactive oxygen species and sustained c-Jun-NH2 kinase signaling. It was interesting that adenoviral vector-mediated Glipr1 (AdGlipr1) transduction into prostate cancer tissues using an immunocompetent orthotopic mouse model revealed additional biologic activities consistent with tumor-suppressor functions. Significantly reduced tumor-associated angiogenesis and direct suppression of endothelial-cell sprouting activities were documented. In addition, AdGlipr1 strongly stimulated antitumor immune responses that resulted in specific cytotoxic T-lymphocyte activities in this model. Glipr1-related antitumor immunostimulatory activities were confirmed and extended in subsequent studies. Administration of a novel Glipr1 gene-modified tumor cell vaccine had significant antitumor activity in a mouse model of recurrent prostate cancer. In conclusion, restoration of GLIPR1 function in prostate cancer cells through GLIPR1 gene-based or GLIPR protein-based delivery methods may provide a safe and effective approach for targeted therapy for a range of malignancies.
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Food allergy is an adverse food reaction as a result of immune mechanisms. In a sensitized individual, food allergens activate mast cells and basophils by binding with IgE present on the cell surface, resulting in the release of chemical mediators and various cytokines to cause various clinical symptoms of food allergy. Sensitization to food allergens can occur in the gastrointestinal tract (class 1 food allergy) or as a consequence of cross reactivity to structurally homologous inhalant allergens (class 2 food allergy). The class 1 food allergens are water-soluble glycoproteins with 10-70 kD size that are resistant to heat, acid and enzymes. On the other hand, the class 2 food allergens are highly unstable and degraded by heat or enzymatic digestion. Much progress has been made in identifying and isolating food allergen. Recently cDNAs for many proteins have been isolated and recombinant proteins have been generated. These techniques make it easier to characterize each responsible food allergens. Plant food allergens are classified into families and superfamilies by their structural and functional properties. The most of plant food allergens are the cupin and prolamin superfamilies and the protein families of the plant defense system. The cupin superfamily includes allergenic seed storage proteins of 7s globulin (vicilin) and 11s globulin (legumin). 2s albumin seed storage proteins, the nonspecific lipid transfer proteins, and the cereal alpha-amylase and protease inhibitors belong to the prolamin superfamily. Profilins, heveins, and nonspecific lipid transfer proteins are present in a variety of pollens, nuts, seeds, fruits, and vegetables. These are considered as panallergens, causing a significant degree of IgE-mediated cross-reactivity. Detailed informations about the character of food allergens can be used to develop more sophisticated diagnostic methods and treatment modalities in the near future. Further knowledge of food allergens is also useful to assess the allergenicity of novel protein of genetically mo.