Fuxiang Bao

Introduction: ATP-binding cassette subfamily D member 4 (ABCD4) is an ATP-binding cassette transporter involved in intracellular cobalamin trafficking and has also been identified as a candidate gene associated with vertebral-number variation in livestock. However, molecular information and experimental resources for ovine ABCD4 remain limited. This study aimed to characterize a RACE-derived ABCD4 cDNA sequence from Ujimqin sheep, analyze its sequence and predicted molecular features, establish a recombinant protein expression system, and generate a polyclonal antibody resource for subsequent studies. Methods: Total RNA isolated from Ujimqin sheep liver was subjected to rapid amplification of cDNA ends (RACE), followed by sequence analysis, phylogenetic comparison, physicochemical characterization, recombinant expression in Escherichia coli, protein purification, and antibody production. Results: A 1,874-bp ABCD4 cDNA sequence was obtained, containing a predicted 1,209-bp open reading frame encoding a 402-amino-acid polypeptide. Sequence and bioinformatic analyses showed that the RACE-derived sequence retained conserved features of the ABC transporter family, although its predicted protein length was shorter than that of canonical mammalian ABCD4 proteins. For recombinant antigen production, the ABCD4 expression construct was introduced into E. coli BL21 (DE3), and higher recombinant protein expression was observed after induction with 0.5 mM IPTG at 37 °C for 4 h. The recombinant protein was predominantly recovered from the insoluble fraction and purified by Ni-NTA affinity chromatography. Purified recombinant protein was then used to immunize New Zealand White rabbits solely for polyclonal antiserum production, and the resulting antisera showed strong immunoreactivity against the recombinant antigen. Discussion: Overall, this study provides a RACE-derived ovine ABCD4 cDNA sequence together with recombinant protein and antibody resources that may support future investigations of ABCD4 expression, localization, and biological function in sheep.

Clostridium perfringens is a major livestock pathogen responsible for histotoxic diseases and intestinal infections, posing serious threats to public health and the poultry and breeding industry. Overreliance on antibiotics has driven the emergence of drug-resistant strains and disruption of gut microbiota homeostasis, underscoring the urgent need for alternative control strategies. In this study, a lytic phage designated vB_CppII_Hohhot was isolated from wastewater samples collected from large-scale dairy farms in Inner Mongolia. The optimal multiplicity of infection was determined to be 0.1. pH stability was assessed. The phage was found to remain stable across pH 3 to 11, but no viable phage was detected at pH 12 (detection limit: C. perfringens-associated infections.

Feline lymphoma, a prevalent feline malignancy, responds poorly to conventional chemotherapy-a setting that underscores the urgent need for targeted therapeutic alternatives. The stable expression of CD3 on T-cell lymphoma cells positions this molecule as an ideal immunotherapeutic target. Compared with conventional antibodies, single-domain nanobodies derived from camelid heavy-chain antibodies offer distinct advantages, including smaller size, enhanced tissue penetration, and superior stability, thereby rendering CD3-targeted nanobodies particularly promising for managing feline T-cell lymphoma. We immunized a Bactrian camel with recombinant feline CD3ε extracellular domain and constructed a phage display nanobody library (capacity 5.97 × 10⁸). Sequencing of the constructed library revealed high diversity; among 10 randomly selected clones, 7 unique sequences were identified, and 93.33% of the clones carried intact VHH inserts. After three panning rounds, three high-affinity single-domain antibodies were selected and expressed, with clone 73 retaining stable binding at 156 ng/mL concentration. Flow cytometry showed that clone 73 specifically recognized approximately 37.95% of feline peripheral blood CD3⁺ cells. Western blotting detected a specific ~ 20 kDa band in feline lymphocyte lysates, and immunofluorescence confirmed precise cellsurface localization without non-specific signal. These findings demonstrate successful generation of feline CD3-specific nanobodies, which enable flow cytometric diagnosis of feline T-cell lymphoma and provide a foundation for developing immunotherapeutics.

To address the antigen display limitations of current vaccine carriers, we engineered the T4 bacteriophage into a high-capacity platform. The T4 bacteriophage has advantages such as structural stability, high loading capacity, and easy production. However, the presence of high-copy endogenous Soc protein on its capsid surface severely restricts the effective display of exogenous proteins. To overcome the aforementioned spatial limitations, we employed the CRISPR/Cas9 system to precisely knockout the Soc gene of T4 bacteriophage. We successfully achieved the knockout of the Soc gene by co-transferring the three plasmid systems-pCas, pTargetF-sgRNA, and pMD19-T-Soc-arm-into Escherichiacoli TG1, inducing the expression of Cas9 with L-arabinose, and then infecting the engineered bacteria with the wild-type T4 bacteriophage (named T4 WT). The PCR, SDS-PAGE, and sequencing results confirmed a Soc gene-deficient T4 bacteriophage mutant strain (named T4ΔSoc) was successfully constructed. This mutant strain had comparable growth, thermal stability, and pH stability to the wild type, and maintained complete infectivity. After continuous passage for five generations, it remained stable in terms of genome, protein composition, and phage plaque phenotype, with no occurrence of revertant mutations. In conclusion, T4ΔSoc is stable both genetically and phenotypically, effectively breaking through the limitation of the restricted display space of T4 WT. This study provides a reliable vector tool and technical foundation for constructing an efficient and stable antigen display and vaccine delivery platform and has good application potential in vaccine research and the construction of targeted delivery systems.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disorder characterized by dysregulated hepatic lipid metabolism, with a continuously rising global incidence and limited safe and effective therapeutic options. Importantly, NAFLD-like conditions, namely hepatic lipidosis or fatty liver syndrome, also prevail in veterinary clinical practice, affecting companion animals (obese cats and dogs) and livestock (periparturient dairy cows, fattening pigs, and broiler chickens). These metabolic liver disorders are primarily induced by inappropriate feeding management and metabolic stress, leading to reduced production performance and survival rate of animals, huge economic losses to the livestock industry, and impaired health of companion animals. Flavonoid compounds derived from traditional Chinese medicine (TCM) possess the advantages of low toxicity and multi-target pharmacological effects, and have emerged as promising natural agents for NAFLD amelioration. Methods: This study adopted a systematic review approach to comprehensively collect, sort out, and summarize recent relevant research findings. We focused on widely reported TCM flavonoids, including quercetin, apigenin, and luteolin, and systematically analyzed the diverse molecular pathways and potential mechanisms by which these compounds exert protective effects against NAFLD and veterinary fatty liver diseases. Results: Existing research evidence demonstrates that TCM flavonoids improve NAFLD through multiple core regulatory pathways. These compounds reverse hepatic lipid metabolism disorders by activating the AMPK/SIRT1 and PPARα signaling pathways, inhibit lipogenesis by suppressing the key lipogenic factor SREBP-1c , and accelerate lipid catabolism by promoting fatty acid β -oxidation. In addition, flavonoids effectively alleviate hepatic oxidative stress, inhibit inflammatory responses, and delay the progression of liver fibrosis. Furthermore, they exert protective effects via regulating novel mechanisms, including cellular autophagy, ferroptosis, and intestinal microbiota homeostasis. Discussion: The multi-target and systematic regulatory characteristics of TCM flavonoids make them excellent candidate natural drugs for NAFLD intervention in both human and veterinary medicine. Nevertheless, several limitations and challenges remain in current research, including low bioavailability of flavonoids and unclear synergistic effects among different flavonoid components. Future studies should focus on improving the bioavailability of flavonoids, elucidating their synergistic molecular mechanisms, and exploring species-specific pharmacokinetic characteristics in cats, dogs, and cattle. Moreover, the development of practical and palatable preparations such as feed additives is essential to promote the clinical translation and large-scale application of flavonoids for the prevention and treatment of NAFLD in human and veterinary clinical practice.

Bovine rotavirus is one of the main pathogens causing acute diarrhea in calves under 1 month of age, posing a serious threat to intensive cattle farming, and there are no effective therapeutic agents owing to the mixed infection of bovine rotavirus (BRV) with other pathogens. In this study, we successfully constructed a phage display antibody library by immunizing Bactrian camels, with a capacity of 6.5 × 10 ⁸ and a recombinant positive rate of 81.25%. Three high-activity variable heavy-chain domains (VHH) were obtained by using BRV as the antigen, and the gene fragments were ligated with the prokaryotic expression vector pET–22b(+). The recombinant VHH was successfully expressed under induction by isopropyl β- d -1-thiogalactopyranoside (IPTG). In addition, the recombinant VHH could specifically bind to BRV in an enzyme-linked immunosorbent assay (ELISA) and cellular immunofluorescence assays. The neutralizing activity of VHH was further confirmed by a virus neutralization test. Furthermore, our data showed that recombinant VHH could alleviate symptoms such as diarrhea and high fever, and reduce the amount of virus release in a BRV-challenged calf model. These results suggest that the single-domain antibodies against BRV screened in this study offer an effective treatment in BRV infection, facilitating the development of novel therapeutics for BRV.

Bovine Viral Diarrhea Virus (BVDV), the etiologic agent of bovine viral diarrhea, poses substantial economic threats to the cattle industry worldwide. While vaccination represents the cornerstone of preventive strategies, relying primarily on inactivated whole-virus vaccines, continuous viral evolution has rendered these conventional formulations inadequately protective. Bovine ultralong CDR H3 antibodies, characterized by a 70-amino-acid composition, emerge as promising candidates for therapeutic development. However, their effectiveness against BVDV remains largely unexplored. In this study, a phage display library encoding BVDV-specific ultralong CDR H3 antibodies was successfully constructed and subjected to biopanning. Selected high-affinity antibodies were expressed and purified, and showed specific binding to BVDV with an affinity of 312.5 ng/mL and neutralizing activity (IC ₅₀ = 11.72 μg/mL) in vitro . To evaluate therapeutic efficacy in vivo , a BALB/c mouse challenge model was established. We found that antibody treatment significantly reduced viral load and attenuated histopathological damage compared to the control group ( P < 0.05). These results collectively underscore the potential of ultralong CDR H3 antibody platforms as viable candidates for novel BVDV immunotherapeutics and vaccine development.

Canine B-cell lymphoma is a malignant hematologic neoplastic disease, with diffuse large B-cell lymphoma being the predominant subtype. This subtype highly expresses canine CD20 on its surface. Most clinical treatments are chemotherapeutic, with the standard treatment being the CHOP regimen. In immunotherapy, rituximab is effective for treating human B-cell lymphoma. However, its inability to bind natural canine CD20 renders it ineffective in treating canine B-cell lymphoma. The main objective of this study was to establish camel-canine chimeric antibodies and validate their in vitro biological activity. A Bactrian camel was immunized with the CD20 extracellular region peptide. The VHH fragment was amplified using nested PCR, ligated to the phage vector pMECS, and used to construct a phage antibody library with a capacity of 3.4 × 1010. After three rounds of enrichment and screening, 92 clones were selected for phage ELISA. Clones 4, 5, 8, 30, 43 and 46 exhibited high binding affinities for canine CD20. These strains were induced and purified using a prokaryotic expression system to obtain anti-canine CD20 single-domain antibodies. ELISA, Western blotting and flow cytometry analysis showed that the anti-canine CD20 single-domain antibody specifically binds to canine CD20. Cellular immunofluorescence validation revealed that the anti-canine CD20 single-domain antibody specifically binds to CD20 on the surface of Raji cells. The anti-canine CD20 single-domain antibody was subsequently ligated to the canine Fc fragment to construct a camel-canine chimeric antibody. The camel-canine chimeric antibody was expressed in CHO-S cells, and ELISA revealed that the chimeric antibody can specifically bind to the canine CD20 extracellular region polypeptide and the cell surface CD20 in Raji cells. In vitro killing assays revealed that the camel-canine chimeric antibody effectively mediated the targeting of canine peripheral blood mononuclear cells to Raji cells. These results indicate that the camel-canine chimeric antibody has strong anti-canine B-cell lymphoma activity and provide a basis for further development and clinical application of anti-canine B-cell lymphoma drugs.

Bovine rotavirus (BRV) is one of the main pathogens that cause acute diarrhea in calves under one month of age. Passive immunization has been recognized as an effective way to prevent and treat BRV infection. Recent studies have shown that 10% of bovine antibodies possess an ultra-long CDR H3 domain, which has been shown to be the smallest antigen-binding domain. Due to the extremely small size of ultra-long CDR H3 antibodies, the phage display method was utilized to obtain ultra-long CDR H3 antibodies targeting BRV, providing a new approach for the prevention and/or treatment of BRV. Here, we report the preparation of BRV-specific bovine ultra-long CDR H3 antibodies obtained by constructing and screening a phage display library containing approximately 8.55 × 10⁹ individual clones. Through three rounds of bio-panning, we identified 92 candidate clones, of which 79 exhibited specific binding activity in phage ELISAs. The recombinant bovine ultra-long CDR H3 antibodies could specifically bind to BRV in ELISAs and cell immunofluorescence assays. The neutralizing activity was further confirmed through virus neutralization tests. In the calf model experiment, the recombinant bovine ultra-long CDR H3 antibodies could relieve the symptoms of diarrhea, reduce both the amount and duration of virus release, and increase the survival in calves experimentally infected with BRV. Therefore, BRV-specific bovine ultra-long CDR H3 antibodies could serve as an effective agent for the prevention and treatment of BRV infection. At the same time, the development of ultra-long CDR H3 antibodies using phage display screening technology provides a new approach for developing biological agents for the prevention and control of infectious diseases in bovines.