This study aims to establish an ELISA method with high specificity for the detection of antibodies against Mycoplasma hyopneumoniae. Firstly, we constructed a recombinant strain Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 to express the recombinant protein Mhp336 and used the purified Mhp336 as the coating antigen. Then, we optimized the ELISA parameters, including antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, colorimetric reaction time, and cut-off value. Afterwards, reproducibility experiments were conducted, and the cross reactivity of Mhp366 with other antisera of porcine major pathogens and the maximum dilution ratios of the sera were determined. Finally, 226 porcine serum samples were detected using the method established in this study, a commercial ELISA kit for M. hyopneumoniae antibody detection, and a convalescent serum ELISA kit for M. hyopneumoniae antibody detection. The detection results of the three methods were compared to evaluate the sensitivity and specificity of the ELISA method established in this study. For this method, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, and colorimetric reaction time were 0.05 μg/mL, PBS containing 2.5% skim milk, 1 h, 1:500, 0.5 h, 1:10 000, 1 h, and 5 min, respectively. Validation of the ELISA method based on Mhp336 showed a cut-off value of 0.332. The coefficients of variation of both intra-batch and inter-batch kits were below 7%. The detection results of porcine serum samples indicated that the method established in this study outperformed the commercial ELISA kit and the convalescent serum ELISA kit for M. hyopneumoniae antibody detection in terms of sensitivity and specificity. We successfully established an ELISA method for detecting the antibodies against M. hyopneumoniae based on Mhp336 protein. This method demonstrated high sensitivity and specificity, serving as a tool for the prevention of mycoplasmal pneumonia of swine in pig farms.

Tuberculosis (TB) is one of the infectious diseases caused by the pathogen Mycobacterium tuberculosis that continuously threatens the global human health. Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine that has been used clinically to prevent tuberculosis in recent centuries, but its limitations in preventing latent infection and reactivation of tuberculosis do not provide full protection. In this study, we selected the membrane-associated antigen Rv1513 of Mycobacterium. In order to achieve stable expression and function of the target gene, the prokaryotic expression recombinant vector pET30b-Rv1513 was constructed and expressed and purified its protein. Detection of IFN- γ levels in the peripheral blood of TB patients stimulated by whole blood interferon release assay (WBIA) and multi-microsphere flow immunofluorescence luminescence (MFCIA) revealed that the induced production of cytokines, such as IFN-γ and IL-6, was significantly higher than that in the healthy group. Rv1513 combined with adjuvant DMT (adjuvant system liposomes containing dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL), and trehalose-660-dibenzoic acid (TDB)) was used to detect serum specific antibodies, cytokine secretion from splenic suprasplenic cell supernatants, and multifunctional T-cell levels in splenocytes in immunised mice. The levels of IFN-γ, TNF-α, and IL-2 secreted by mouse splenocytes were found in the Rv1513+DMT group and the BCG+Rv1513+DMT group. The serum levels of IgG and its subclasses and the number of IFN-γ+T cells, TNF-α+T and IFN-γ+TNF-α+T cells in the induced CD4+/CD8+T cells in mice were significantly higher than those in the BCG group, and the highest levels were found in the BCG+Rv1513+DMT group. These findings suggest that Rv1513/DMT may serve as a potential subunit vaccine candidate that may be effective as a booster vaccine after the first BCG vaccination.

The antimicrobial peptide LRGG (LLRLLRRGGRRLLRLL-NH2) was designed and chemically synthesized in a study conducted by Jia et al. Gram-negative bacteria were found to be sensitive to LRGG and exhibited a high therapeutic index. Genetic engineering methods were used to create the prokaryotic fusion expression vector pQE-GFP-LRGG, and the resulting corresponding fusion protein GFP-LRGG was subsequently expressed and purified. The precursor GFP was then removed by TEV proteolysis, and pure LRGG was obtained after another round of purification and endotoxin removal. The prokaryotic-expressed antimicrobial peptide LRGG displays a broad-spectrum antibacterial effect on Gram-negative bacteria, and its minimum inhibitory activity (MIC) against Escherichia coli can reach 2 μg/mL. Compared to the chemically synthesized LRGG, the prokaryotic-expressed LRGG exhibits similar temperature, pH, salt ion, serum stability, and cell selectivity. Furthermore, prokaryotic-expressed LRGG showed excellent therapeutic effects in both the infection model of cell selectivity and no embryotoxicity in a Galleria mellonella infection model. The mechanism by which LRGG causes bacterial death was found to be the disruption of the Gram-negative cell membrane.

BACKGROUND: The neuroimmune network plays a crucial role in regulating mucosal immune homeostasis within the digestive tract. Synaptosome-associated protein 25 (SNAP-25) is a presynaptic membrane-binding protein that activates ILC2s, initiating the host\'s anti-parasitic immune response.
METHODS: To investigate the effect of Moniezia benedeni (M. benedeni) infection on the distribution of SNAP-25 in the sheep\'s small intestine, the recombinant plasmid pET-28a-SNAP-25 was constructed and expressed in BL21, yielding the recombinant protein. Then, the rabbit anti-sheep SNAP-25 polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of SNAP-25 in the intestines of normal and M. benedeni-infected sheep were detected by ELISA.
RESULTS: The results showed that the SNAP-25 recombinant protein was 29.3 KDa, the titer of the prepared immune serum reached 1:128,000. It was demonstrated that the rabbit anti-sheep SNAP-25 polyclonal antibody could bind to the natural protein of sheep SNAP-25 specifically. The expression levels of SNAP-25 in the sheep\'s small intestine revealed its primary presence in the muscular layer and lamina propria, particularly around nerve fibers surrounding the intestinal glands. Average expression levels in the duodenum, jejunum, and ileum were 130.32 pg/mg, 185.71 pg/mg, and 172.68 pg/mg, respectively. Under conditions of M. benedeni infection, the spatial distribution of SNAP-25-expressing nerve fibers remained consistent, but its expression level in each intestine segment was increased significantly (P < 0.05), up to 262.02 pg/mg, 276.84 pg/mg, and 326.65 pg/mg in the duodenum, jejunum, and ileum, and it was increased by 101.06%, 49.07%, and 89.16% respectively.
CONCLUSIONS: These findings suggest that M. benedeni could induce the SNAP-25 expression levels in sheep\'s intestinal nerves significantly. The results lay a foundation for further exploration of the molecular mechanism by which the gastrointestinal nerve-mucosal immune network perceives parasites in sheep.

Since 2017, an infectious goose gout disease characterized by urate precipitation in viscera, mainly caused by novel goose astrovirus (GoAstV) infection, has emerged in the main goose-producing region of China. The current challenge in managing goose gout disease is largely due to the absence of a rapid and efficient detection method for the GoAstV pathogen. Notably, the potential application of immunosensors in detecting GoAstV has not yet been explored. Herein, a label-free PEC immunosensor was fabricated by using purchased TiO2 as the photoactive material and antibody against GoAstV P2 proteins as the specific recognition element. First, we successfully expressed the capsid spike domain P2 protein of ORF2 from GoAstV CHSH01 by using the pET prokaryotic expression system. Meanwhile, the polyclonal antibody against GoAstV capsid P2 protein was produced by purified protein. To our knowledge, this is the first establishment and preliminary application of the label-free photoelectrochemical immunosensor method in the detection of AstV. The PEC immunosensor had a linear range of 1.83 fg mL-1 to 3.02 ng mL-1, and the limit of detection (LOD) was as low as 0.61 fg mL-1. This immunosensor exhibited high sensitivity, great specificity, and good stability in detecting GoAstV P2 proteins. To evaluate the practical application of the immunosensor in real-world sample detection, allantoic fluid from goose embryos was collected as test samples. The results indicated that of the eight positive samples, one false negative result was detected, while both negative samples were accurately detected, suggesting that the constructed PEC immunosensor had good applicability and practical application value, providing a platform for the qualitative detection of GoAstV.

The functions of highly expressed genes DFP1 and DFP2 in Dermatophagoides farinae remain unknown. DFP1 and DFP2 have been abundantly annotated and were up-regulated under temperature stress at 43 °C and -10 °C in our previous RNA-seq study, indicating that DFP1 and DFP2 may have temperature stress response function. Here, we amplified, cloned, and sequenced to obtain the complete coding sequences of DFP1 and DFP2 and predicted their protein characteristics using bioinformatics analysis. Then, prokaryotic expression systems were constructed and found that DFP1 was expressed in Escherichia coli Rosetta-gami 2 (DE3) but not BL21 (DE3); DFP2 was expressed in both BL21 (DE3) and Rosetta-gami 2 (DE3), with higher expression in BL21 (DE3). Finally, the growth curves of bacteria were drawn and indicated that the DFP1- and DFP2-pET32a carrying recombinant bacteria grew better than the respectiveonly pET32a carrying control bacteria after heat and cold stress. This study confirms for the first time that DFP1 and DFP2 respond to temperature stress at the protein level. The constructed prokaryotic expression systems will provide an experimental foundation for future antibody preparation for western blotting detection to confirm the temperature-stress response functions of DFP1 and DFP2.

Fructose 1,6-bisphosphate aldolase (FBA) is a pivotal enzyme, which plays a critical role in fixing CO2 through the process of in the Calvin cycle. In this study, a comprehensive exploration of the FBA family genes in moso bamboo (Phyllostachys edulis) was conducted by the bioinformatics and biological analyses. A total of nine FBA genes (PeFBA1-PeFBA9) were identified in the moso bamboo genome. The expression patterns of PeFBAs across diverse tissues of moso bamboo suggested that they have multifaceted functionality. Notably, PeFBA8 might play an important role in regulating photosynthetic carbon metabolism. Co-expression and cis-element analyses demonstrated that PeFBA8 was regulated by a photosynthetic regulatory transcription factor (PeGLK1), which was confirmed by yeast one-hybrid and dual-luciferase assays. In-planta gene editing analysis revealed that the edited PeFBA8 mutants displayed compromised photosynthetic functionality, characterized by reduced electron transport rate and impaired photosystem I, leading to decreased photosynthesis rate overall, compared to the unedited control. The recombinant protein of PeFBA8 from prokaryotic expression exhibited enzymatic catalytic function. The findings suggest that the expression of PeFBA8 can affect photosynthetic efficiency of moso bamboo leaves, which underlines the potential of leveraging PeFBA8\'s regulatory mechanism to breed bamboo varieties with enhanced carbon fixation capability.

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb\'s as IgG2b and its light chain as κ-type; the 5E9 mAb\'s heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody\'s application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbβ3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.