Pacific abalone (Haliotis discus hannai) is a marine gastropod mollusc with significant economic importance in both global fisheries and aquaculture. However, studies exploring the gonadal development and regulatory mechanisms of Haliotis discus hannai are limited. This study aimed to explore whether the vasa gene acted as a molecular marker for germ cells. Initially, the vasa gene was successfully cloned using the cDNA-end rapid amplification technique. The cloned gene had a 2478-bp-long open reading frame and encoded 825 amino acids. Then, a recombinant expression vector was constructed based on the Vasa protein, and an 87-kDa recombinant protein was prepared. Subsequently, a polyclonal antibody was prepared using the purified recombinant protein. The enzyme-linked immunosorbent assay (ELISA) confirmed the titer of the antibody to be ≥512 K. The immunohistochemical analysis revealed that Vasa was widely expressed in oogonia, Stage I oocytes, spermatogonia, and primary spermatocytes. The specific expression of Vasa in the hermaphroditic gonads of abalone was assessed using western blotting to investigate the effects of different photoperiods (12 L:12D, 24 L:0D, 18 L:6D, and 6 L:18D) on the gonadal development of abalone (P < 0.05), with higher expression levels observed in the ovarian proliferative and spermary maturing stages compared with other developmental stages (P < 0.05). Additionally, Vasa exhibited the highest expression in the spermary and ovary under a photoperiod of 18 L:6D (P < 0.05). These data demonstrated the key role of Vasa in developing germ cells in abalone. They shed light upon the molecular mechanism through which the photoperiod influenced Vasa expression and regulated gonadal development in abalone. The findings might provide theoretical references for analyzing the differentiation pattern of abalone germ cells and the genetic improvement and conservation of germplasm resources.

OBJECTIVE: To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications.
METHODS: The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA).
RESULTS: SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells.
CONCLUSIONS: The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.
［摘要］ 目的 制备并鉴定鼠抗刚地弓形虫致密颗粒蛋白24 (dense granule protein 24, GRA24) 多克隆抗体, 并探索其初 步应用。方法 利用MEGA-X软件比对弓形虫不同虫株GRA24编码区序列, 使用Protean软件分析GRA24蛋白优势肽 段, 通过PCR反应扩增编码该肽段的碱基序列, 并连接至pET-28a载体中。将获得的GRA24截短蛋白原核表达质粒转化 于大肠埃希菌BL21感受态细胞中, 异丙基-β-D-硫代半乳糖苷 (isopropyl-beta-D-thiogalactopyranoside, IPTG) 诱导后采用 十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 检测蛋白表达 与纯化。使用纯化的GRA24截短蛋白皮下注射免疫BALB/c小鼠获得GRA24截短蛋白多克隆抗体, 采用酶联免疫吸附 试验 (enzyme-linked immunosorbent assay, ELISA) 检测抗体效价, 采用Western blotting检测抗体特异性, 并将该抗体应用 于免疫荧光试验 (immunofluorescence assay, IFA) 。结果 SDS-PAGE结果表明成功构建重组质粒, 考马斯亮蓝染色结果 显示获得高纯度重组GRA24截短蛋白。ELISA结果显示, GRA24 截短蛋白多克隆抗体效价在 1:208 400 以上; Western blotting检测发现, 该抗体可识别弓形虫不同虫株内源性 GRA24 蛋白, 特异性识别重组 GRA24 截短蛋白; 间接IFA检测发 现, 弓形虫入侵宿主细胞 16 h 后分泌的GRA24蛋白定位于宿主细胞核中。结论 成功制备广适性、高效价、强特异性的 抗弓形虫 GRA24 多克隆抗体, 可应用于 Western blotting 与 IFA, 为进一步研究 GRA24 功能奠定了基础。.

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.

Cell migration regulated by Thrombospondin 2 (THSB2) is important for the development of pulmonary artery remodeling, but the mechanism by which THBS2-mediated cell migration regulates the development of pulmonary artery remodeling in broiler ascites syndrome (AS) is unclear. In addition, the lack of chicken THBS2 antibodies makes it difficult to study the mechanism in depth. In our study, we used recombinant gene technology, protein purification, and other techniques to obtain mouse anti-chicken THBS2 antibody and analyze its expression in broilers, ascites broilers and other animals. The results showed that we immunized mouse with recombinant THBS2 protein and obtained an antibody titer of 1:204,800, and the addition of astragalus polysaccharide as an immunomodulator during immunization significantly increased the titer of the antibody. Western blotting (WB) and immunofluorescence results showed that the THBS2 was significantly down-regulated in the ascites broiler. The THBS2 antibody we prepared can also detect THBS2 protein in duck, mouse, goat, and rabbit tissues. These results provide a foundation for further investigation of the role of THBS2 in pulmonary artery remodeling in broiler ascites syndrome and a powerful tool for studying the role of THBS2 in AS.

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL-1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL-1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

In recent years, the nephrotoxicity and carcinogenicity of aristolochic acid have attracted worldwide attention, and the traditional Chinese medicine containing this ingredient has been banned in many places, affecting the TCM industry. To meet this challenge, researchers have developed various detection methods, such as high-performance liquid chromatography, gas chromatography-mass spectrometry and thin-layer chromatography. A rapid detection method must therefore be developed to ensure safety. A polyclonal antibody capable of recognizing aristolochic acid was prepared, and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established to detect the amount of aristolochic acid in the sample to be measured. Methods Using 1-(4-chlorophenyl) cyclobutylamine as a hapten, immunogens and coating antigens were obtained by coupling with bovine serum albumin (BSA) and chicken ovalbumin (OVA) using the active ester method. UV scanning confirmed the successful coupling of the conjugate, and New Zealand white rabbits were immunized. The obtained antibody serum was screened for the best antibody by ic-ELISA detection. Use the chessboard method to determine three optimal combinations of original coating concentration and antibody dilution ratio, establish a standard curve for each combination to obtain the best combination, and establish a rapid detection method. Finally, the standard aristolochic acid A was added to the purchased apple vinegar and canned coffee for recycling experiments to verify the detection method.By changing the antigen antibody concentration, the antibody showed the highest sensitivity to aristolochic acid standard at the original coating, 1000-fold dilution, IC50 of 24.88 ng/mL, limit of detection IC10 of 3.19 ng/mL, and detection range IC20-IC80 of 6.81-90.91 ng/mL. The recovery experiments under this conditions yielded a recovery rate of 92%-105%, within reasonable limits, indicating the success of the ELISA rapid detection method. Conclusion The enzyme-linked immunoassay method established in this paper can quickly detect the content of aristolochic acid in the sample to be tested, and the antibody prepared by this method has good broad-spectrum and can detect other aristolochic acid, such as aristolochic acid A, aristolochic acid B, aristolochic acid C, and aristolochic acid D.

p62, also known as SQSTM1, has been shown to be closely related to the coronavirus. However, it remains unclear on the relationship between p62 and NIBV infection. Moreover, there are no available antibodies against the chicken p62 protein. Thus, this study aimed to prepare p62 polyclonal antibody and investigate the correlation between the p62 protein and NIBV infection. Here, PET-32a-p62 prokaryotic fusion expression vector was constructed for prokaryotic protein expression, and then p62 polyclonal antibody was prepared by immunizing rabbits. Lastly, these antibodies were then utilized in Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) assays. The results showed that we successfully prepared chicken p62 polyclonal antibody. Meanwhile, WB and IF demonstrated that the expression of p62 showed a trend of first increase and then decrease after NIBV infection. IHC showed that the expression of p62 in the spleen, lung, kidney, bursa of Fabricius and trachea of chickens infected with NIBV in 11 dpi was significantly higher than that of normal chickens. Taken together, this study successfully prepared a polyclonal antibody for chicken p62 protein and confirmed its application and expression in chickens, as well as the expression of p62 in tissues after NIBV infection.

The interleukin 23 receptor (IL-23R) is associated with a variety of inflammatory diseases in humans and other mammals. However, whether IL-23R is involved in inflammatory diseases in teleost fish is less understood. Thus, to investigate the potential involvement of IL-23R in fish inflammatory diseases, the full-length cDNA of IL-23R from grass carp Ctenopharyngodon idella was cloned and used to generate a recombinant protein (rgcIL-23R) containing the extracellular domain of IL-23R, against which a polyclonal antibody (rgcIL-23R pAb) was then developed. qPCR analysis revealed that IL-23R mRNA was significantly upregulated in most grass carp tissues in response to infection with Gram-negative Aeromonas hydrophila. Treatment with rgcIL-23R significantly induced IL-17A/F1 expression in C. idella kidney (CIK) cells. By contrast, knockdown of IL-23R caused significant decreases in IL-23R, STAT3, and IL-17N expression in CIK cells after lipopolysaccharide (LPS) stimulation. Similarly, rgcIL-23R pAb treatment effectively inhibited the LPS-induced increase in the expression of IL-23 subunit genes and those of the IL-23/IL-17 pathway in CIK cells. Furthermore, intestinal symptoms identical to those caused by A. hydrophila were induced by anal intubation with rgcIL-23R, but suppressed by rgcIL-23R pAb. Therefore, these results suggest that IL-23R has a crucial role in the regulation of intestinal inflammation and, thus, is a promising target for controlling inflammatory diseases in farmed fish.

Neosporosis and toxoplasmosis are major causes of abortion in livestock worldwide, leading to substantial economic losses. Detection tools are fundamental to the diagnosis and management of those diseases. Current immunohistochemistry (IHC) tests, using sera raised against whole parasite lysates, have not been able to distinguish between Toxoplasma gondii and Neospora caninum. We used T. gondii and N. caninum recombinant proteins, expressed in Escherichia coli and purified using insoluble conditions, to produce specific polyclonal rabbit antisera. We aimed to develop species-specific sera that could be used in IHC on formalin-fixed, paraffin-embedded (FFPE) tissue sections to improve the diagnosis of ruminant abortions caused by protozoa. Two polyclonal rabbit sera, raised against recombinant proteins, anti-Neospora-rNcSRS2 and anti-Toxoplasma-rTgSRS2, had specificity for the parasite they were raised against. We tested the specificity for each polyclonal serum using FFPE tissue sections known to be infected with T. gondii and N. caninum. The anti-Neospora-rNcSRS2 serum labeled specifically only N. caninum-infected tissue blocks, and the anti-Toxoplasma-rTgSRS2 serum was specific to only T. gondii-infected tissues. Moreover, tissues from 52 cattle and 19 sheep previously diagnosed by lesion profiles were tested using IHC with our polyclonal sera and PCR. The overall agreement between IHC and PCR was 90.1% for both polyclonal anti-rNcSRS2 and anti-rTgSRS2 sera. The polyclonal antisera were specific and allowed visual confirmation of protozoan parasites by IHC, but they were not as sensitive as PCR testing.

UNASSIGNED: To obtain a reactive and specific antibody against truncated matrix metalloproteinase 9 (MMP-9), that has reactivity toward the native protein. Precision, accuracy, specificity, and sensitivity were evaluated using a point-of-care test.
UNASSIGNED: An in silico study was used to confirm the anti peptide truncated MMP-9 is against native MMP-9. After an antibody titer assessment, purification, and characterization, the anti MMP-9 was assessed. The cut-off value was determined using the purified gelatinases of the supernatant HCT 116 cell line. The supernatant was purified by preparative native-polyacrylamide gel electrophoresis based on charge and size of the proteins. Furthermore, quality control (QC) of the results were performed following standard densitometry methods.
UNASSIGNED: A truncated MMP-9 is the major epitope peptide that can trigger the immune system to scavenge for a specific and reactive antibody against the native MMP-9. The MMP-9 native protein is purified from the supernatant of the HCT 116 cell line and the commercially available, full-length MMP-9. The cut-off value was estimated at 30 μg/mL. QC results indicated that the specificity was 80%, sensitivity was 96.7%, accuracy was 91%, and precision was 91.66%. The area under curve was 0.827 (P < 0.001). The positive predictive value was 83%, and the negative predictive value was 96%.
UNASSIGNED: The antibody against the synthetic epitope peptide can detect the native MMP-9. Native MMP-9 is considered the main biomarker in an immunoassay POCT and is used to diagnose dry eye disease (DED). In accordance with QC results, MMP-9 point of care test can be utilized for screening patients suffering from DED.