Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.
Animals ; Baculoviridae/metabolism* ; Bass/metabolism* ; Carps/virology* ; Cell Line ; Female ; Genetic Techniques ; Genome, Viral ; Glycoproteins/biosynthesis* ; Insecta ; Ovary/virology* ; Phylogeny ; Plasmids/metabolism* ; Recombinant Proteins/biosynthesis* ; Rhabdoviridae/metabolism*

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.
Adolescent ; Adult ; Allergens/*chemistry/*immunology ; Amino Acid Sequence ; Animals ; Bombyx/*chemistry/genetics/growth & development/*immunology ; Epitopes/immunology ; Female ; Food Hypersensitivity/etiology ; Glycoproteins/*chemistry/genetics/*immunology ; Hot Temperature ; Humans ; Immunoglobulin E/immunology ; Male ; Molecular Sequence Data ; Molecular Weight ; Proteomics ; Pupa/chemistry/immunology ; Recombinant Proteins/biosynthesis/chemistry/immunology ; Sequence Alignment

Pituitary adenomas (PAs) are well known as a common intracranial benign tumor, and a portion of PAs are refractory to current therapeutic methods. ErbB receptors family signaling pathway regulates the expression of PAs activation associated gene. Inhibition of epidermal growth factor receptor (EGFR) can inhibit proliferation of PAs. Leucine-rich repeats and immunoglobulin-like domains protein 1 ( LRIG1), a negative mediated gene of ErbB receptors family, plays a role in many tumors. However, there are seldom researches about the functional role of LRIG1 in PAs. The aim of this study is to explore the potential effect of LRIG1 and its regulating mechanism in PAs. First, we investigated the role of LRIG1 in cell migration, invasion of PAs with transfected LRIG1 or control. Then, we explored its impact on cell proliferation and apoptosis of PAs in vivo. To study the regulating mechanism of LRIG1, we examined the expression of molecular factor of PI3K/AKT and Ras/Raf/ERK pathway using Western blotting in vitro and RT-PCR in vitro and in vivo. It was found that LRIG1 over-expression inhibited cell migration, invasion and proliferation, and promoted apoptosis of PAs in vivo and in vitro. Furthermore, LRIG1 suppressed the expression of signaling of PI3K/AKT and Ras/Raf/ERK pathways in PAs. LRIG1, as a negative mediated gene of tumor, can inhibit biological function of PAs via inhibiting PI3K/AKT and Ras/Raf/ERK pathways, and it might be a new target for gene therapy of PAs.
Animals ; Apoptosis ; genetics ; Brain Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MAP Kinase Signaling System ; genetics ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; Oncogene Protein v-akt ; biosynthesis ; Phosphatidylinositol 3-Kinases ; genetics ; Pituitary Neoplasms ; genetics ; pathology ; Xenograft Model Antitumor Assays ; raf Kinases ; biosynthesis ; genetics

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.
Animals ; Codon ; Electrophoresis, Polyacrylamide Gel ; Endonucleases ; biosynthesis ; Glycoproteins ; Hot Temperature ; Penaeidae ; enzymology ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis

The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.
Coprinus ; enzymology ; Culture Media ; Cytoplasm ; Fermentation ; Glycoproteins ; metabolism ; Mass Spectrometry ; Mating Factor ; Oxidoreductases Acting on Sulfur Group Donors ; metabolism ; Peptides ; Peroxidases ; biosynthesis ; Pichia ; metabolism ; Protein Disulfide-Isomerases ; metabolism ; Protein Folding ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins ; metabolism

OBJECTIVE: In this study, we have detected the expression of STC2 protein in laryngeal cancer and The safe incisal margin by immunohistochemical staining, to evaluate its correlation with clinical features and the prognosis value in laryngeal squamous cell cancer. METHOD: Eighty-one paraffin-embedd specimens of patients who underwent surgery for laryngeal cancer were collected. Clinical data, including date of birth, gender, tumor subsite, tumor stage, RT, and survival status have been recorded. The samples including laryngeal cancer tissues and the safe incisal margin were analyzed for the expression of STC2 protein by immunohistochemical staining. We analyzed the correlations with STC2 expression level and clinical data with software SPSS 18. 0. RESULT: (1)fifty-six cases in 81 cases (69. 1 %) are positive. In 30 cases of the safe incisal margin tissues, STC2 is not expressed, which shows statistically significant differences between the two groups (P<0. 05). (2)Overexpression of STC2 in laryngeal cancer were correlated with the tumor clinical stage and N stage, T stage, tumor site location and histological grade (P<0. 05), but not with genderor age (P> 0. 05). (3)The result showed that the 3-year survival rate is 74. 1%, according to the survival analysis by STC2, STC2 positive group has poorer outcome versus the negative group. (4)The clinical stage displayed the independent value in predicting the tumor prognosis of laryngeal cancer (P<0. 05), not the expression level of STC2. CONCLUSION Overexpression of STC2 in laryngeal cancer patients is correlated with poor outcome, which means STC2 positive group has poorer prognosis than the STC2 negative group.
Carcinoma, Squamous Cell ; metabolism ; mortality ; Glycoproteins ; biosynthesis ; Head and Neck Neoplasms ; metabolism ; mortality ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Laryngeal Neoplasms ; metabolism ; mortality ; Prognosis ; Squamous Cell Carcinoma of Head and Neck ; Survival Analysis ; Survival Rate

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

The present study was designed to target fish for potential bioactive components contained in a Huang Lian Jie Du decoction (HLJDD) and identify the underlying mechanisms of action for the treatment of sepsis at the molecular level. he bioactive components database of HLJDD was constructed and the sepsis-associated targets were comprehensively investigated. The 3D structures of the PAFR and TXA2R proteins were established using the homology modelling (HM) method, and the molecular effects for sepsis treatment were analysed by comparing the bioactive components database and the sepsis targets using computational biology methods. The results of the screening were validated with biological testing against the human oral epidermal carcinoma cell line KB in vitro. We found that multiple bioactive compounds contained in the HLJDD interacted with multiple targets. We also predicted the promising compound leads for sepsis treatment, and the first 28 compounds were characterized. Several compounds, such as berberine, berberrubine and epiberberine, dose-dependently inhibited PGE2 production in human KB cells, and the effects were similar in the presence or absence of TPA. This study demonstrates a novel approach to identifying natural chemical compounds as new leads for the treatment of sepsis.
Anti-Inflammatory Agents, Non-Steroidal ; pharmacokinetics ; Berberine ; analogs & derivatives ; pharmacokinetics ; Dinoprostone ; biosynthesis ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Humans ; KB Cells ; Platelet Membrane Glycoproteins ; drug effects ; Protein Transport ; Receptors, G-Protein-Coupled ; drug effects ; Receptors, Thromboxane A2, Prostaglandin H2 ; drug effects ; Sepsis ; drug therapy ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacokinetics

Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.
Blotting, Western ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Membrane Glycoproteins ; immunology ; Pichia ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; Single-Chain Antibodies ; biosynthesis