Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans

Carbamate pesticides, a new type of broad-spectrum pesticides for controlling pests, mites, and weeds, are developed to address the shortcomings of organochlorine and organophosphorus pesticides. Their widespread use and slow degradation have led to environmental pollution, causing damage to ecosystems and human health. Managing pesticide residues is a pressing issue in the current environmental protection. This study aims to investigate the expression of SyEst870, a member of the SGNH/GDSL hydrolase family in Sphingobium yanoikuyae, in a prokaryotic system and evaluate the ability of the recombinant protein to degrade carbamate pesticides. The prokaryotic expression vector pET-32a-SyEst870 was constructed and transformed into the Escherichia coli BL21 for heterologous expression. The purified protein was studied in terms of enzyme activity and effects of temperature, pH, and metal ions on the enzyme activity, with p-nitrophenol acetate as the substrate and based on the standard curve of p-nitrophenol. LC-MS (liquid chromatography-mass spectrometry) was employed to examine the degradation effects of SyEst870 on carbaryl, metolcarb, and isoprocarb. GC-MS (gas chromatography-mass spectrometry) was employed to detect the degradation products of SyEst870 for the three pesticides. The soluble protein SyEst870 was successfully obtained through the heterologous expression in Escherichia coli, which yielded an enzyme with the activity of 677.5 U after affinity chromatography. SyEst870 exhibited degradation rates of 82.34%, 84.43%, and 92.87% for carbaryl, metolcarb, and isoprocarb, respectively, at an initial concentration of 100 mg/L within 24 h at 30 ℃ and pH 7.0. The primary degradation products of carbaryl were identified as α-naphthol and methyl isocyanate. Metolcarb was mainly degraded into m-cresol and methyl isocyanate, and isoprocarb was mainly degraded into 2-isopropylphenol and methyl isocyanate. Compared with the half-life of carbamate pesticides in the natural environment, which ranges from a few days to several weeks, the recombinant protein SyEst870 can rapidly eliminate the residues of carbamate pesticides. This study lays a foundation for addressing pesticide residues in the environment and in fruits and vegetables.
Escherichia coli/metabolism* ; Sphingomonadaceae/genetics* ; Recombinant Proteins/metabolism* ; Biodegradation, Environmental ; Esterases/metabolism* ; Pesticides/isolation & purification* ; Carbamates/isolation & purification*

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.
Animals ; CTLA-4 Antigen ; genetics ; Cell Proliferation ; HEK293 Cells ; Humans ; Lung Neoplasms ; metabolism ; Programmed Cell Death 1 Receptor ; genetics ; Protein Binding ; Protein Domains ; genetics ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.
Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Hydrogen Sulfide ; metabolism ; Plasmids ; genetics ; Seedlings ; metabolism

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.
Escherichia coli ; genetics ; Glucosides ; chemistry ; Humans ; Protein Domains ; Protein Stability ; Pyrophosphatases ; chemistry ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; chemistry ; isolation & purification ; TRPM Cation Channels ; chemistry ; isolation & purification ; Thrombin ; metabolism

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
Antibodies, Monoclonal ; chemistry ; isolation & purification ; metabolism ; Antibody Specificity ; DNA-Binding Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Humans ; Peptides ; Recombinant Fusion Proteins ; genetics ; metabolism

Pectobacterium carotovorum subsp. carotovorum is one of the world's top ten plant pathogens, mainly infecting cruciferous economic crops and ornamental flowers. In this study, an antibacterial gene cpxP (Gene ID: 29704421) was cloned from the genome of Pectobacterium carotovorum subsp. carotovorum, and constructed on the prokaryotic expression plasmid pET-15b, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), then stability and bacteriostatic experiments of the purified CpxP protein were performed. The final concentration of IPTG was 1 mmol/L, obtaining high-efficiency exogenous expression of the CpxP protein. There was no other protein after purification, and the destined protein exhibited good thermal stability and pH stability. The antibacterial test results showed that the inhibition rate of the CpxP protein on carrot slice was 44.89% while the inhibition rate on potato slice was 59.41%. To further explain its antibacterial mechanism, studying the spatial structure of this protein can provide new ideas for the control of soft rot and new protein pesticide targets.
Anti-Bacterial Agents ; pharmacology ; Bacteria ; drug effects ; Bacterial Proteins ; isolation & purification ; pharmacology ; Escherichia coli ; genetics ; Membrane Proteins ; isolation & purification ; pharmacology ; Pectobacterium carotovorum ; genetics ; metabolism ; Plasmids ; genetics

No abstract available.
Anti-Bacterial Agents/*pharmacology ; Azabicyclo Compounds/*pharmacology ; Bacterial Proteins/genetics ; Ceftazidime/*pharmacology ; Cephalosporins/*pharmacology ; DNA, Bacterial/genetics/metabolism ; Drug Resistance, Bacterial/*drug effects ; Gram-Negative Bacteria/drug effects/*isolation & purification ; Humans ; Microbial Sensitivity Tests ; Penicillanic Acid/*analogs & derivatives/pharmacology ; Pseudomonas aeruginosa/drug effects/isolation & purification ; Real-Time Polymerase Chain Reaction

No abstract available.
Coronavirus Infections/*diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; Nasopharynx/virology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

To determine the lysis spectrum of Yersinia enterocolitica bacteriophage phiYe-F10 and to analyze the relationship between the lysis ability of phiYe-F10 and the virulence gene of Yersinia enterocolitica. To observe the lysis ability of the phage phiYe-F10 to the different Yersinia strains with the double-layer technique. The strains used in this study including 213 of Yersinia enterocolitica and 36 of Yersinia pseudotuberculosis and 1 of Yersinia pestis. The virulence genes of these Yersinia enterocolitica (attachment invasion locus (ail) and enterotoxin (ystA, ystB) and yersinia adhesin A (yadA), virulence factor (virF), specific gene for lipopolysaccharide O-side chain of serotype O : 3 (rfbc) were all detected. Among the 213 Yersinia enterocolitica, 84 strains were O : 3 serotype (78 strains with rfbc gene), 10 were serotype O : 5, 13 were serotype O : 8, 34 were serotype O : 9 and 72 were other serotypes. Of these, 77 were typical pathogenic Yersinia enterocolitica harboring with virulence plasmid (ail+, ystA+, ystB-, yadA+, virF+), and 15 were pathogenic bacterial strains deficiency virulence plasmid (ail+, ystA+, ystB-, yadA-, virF-) and the rest 121 were non pathogenic genotype strains. PhiYe-F10 lysed the 71 serotype O : 3 Yersinia enterocolitica strains which were all carried with rfbc+, including 52 pathogenic Yersinia enterocolitica, 19 nonpathogenic Y. enterocolitica. The phiYe-F10 can not lysed serotype O : 5, O : 9 and other serotype Y. enterocolitica, the lysis rate of serotype O : 3 was as high as 84.5%. The phiYe-F10 can not lysed Yersinia pseudotuberculosis and Yersinia pestis. Yersinia phage phiYe-F10 is highly specific for serotype O : 3 Yersinia enterocolitic at 25 degrees C, which showed a typical narrow lysis spectrum. Phage phiYe-F10 can lysed much more pathogenic Y. enterocolitica than nonpathogenic Y. enterocolitica.
Bacterial Proteins ; genetics ; metabolism ; Bacteriophages ; genetics ; isolation & purification ; physiology ; Host Specificity ; Virulence Factors ; genetics ; metabolism ; Yersinia enterocolitica ; genetics ; metabolism ; virology