Many poxviruses are significant zoonotic pathogens threatening public health. Autophagy, a regulated process vital for cellular homeostasis, can participate in defense against virus invasion. However, the relationship between poxviruses and host cell autophagy is not fully understood. This study shows that vaccinia virus (VACV) induces autophagy but blocks autophagosome-lysosome fusion. Modified vaccinia virus Ankara (MVA), an attenuated VACV strain that cannot replicate in most mammalian cells, fails to do so. Both pharmacological inhibition of early autophagy via 3-MA treatment and genetic ablation of ATG3 and ATG7 led to a significant enhancement of MVA replication. The VACV protein A52 inhibits autolysosome formation by disrupting interactions between SNAP29, STX17, and VAMP8, which is crucial for autophagic flux. Importantly, A52 also promotes the degradation of SNAP29, thereby aiding viral replication. Furthermore, SNAP29 is a newly identified host restriction factor for MVA, as its suppression enables MVA replication in human cells. These findings elucidate how poxviruses modulate autophagy for their own replication and further explain MVA's restriction in human cells.

BackgroundMicroRNAs, a class of small noncoding RNAs, serve as post-transcriptional regulators of gene expression and are present in a stable and quantifiable form in biological fluids. MicroRNAs may influence intra-articular responses and the course of disease, but very little is known about their temporal changes in osteoarthritis.ObjectivesTo identify miRNAs and characterise the temporal changes in their abundance in SF from horses with experimentally induced osteoarthritis. We hypothesised that the abundance of miRNA would change during disease progression.Study designIn vivo experiments.MethodsRNA extracted from synovial fluid obtained sequentially (Day 0, 28 and 70) from nine horses with experimentally induced osteoarthritis was subjected to small RNA sequencing using the Illumina Hiseq 4000 sequencing platform. Differentially abundant miRNAs underwent further validation and mapping of temporal abundance (Day 0, 14, 17, 21, 28, 35, 42, 49, 56, 63 and 70 days after osteoarthritis induction) by microfluidic reverse transcription quantitative real-time PCR. Bioinformatic analyses were performed to predict potential biological associations and target genes of the differentially abundant microRNAs.ResultsSmall RNA sequencing revealed 61 differentially abundant microRNAs at an early osteoarthritis stage (Day 28), and subsequent reverse transcription quantitative real-time PCR analysis validated 20 of these. Significant biological functions of the differentially abundant microRNAs were apoptosis, necrosis, cell proliferation and cell invasion. Following validation, four microRNAs (miRNA-199b-3p, miRNA-139-5p, miRNA-1839 and miRNA-151-5p) were detected in more than 50% of the synovial fluid samples and had higher abundance in osteoarthritic than in control joints.Main limitationsLimited sample size.ConclusionThis is the first study to determine longitudinal changes in synovial fluid microRNA abundance in an equine model of osteoarthritis. Larger studies are needed in naturally occurring osteoarthritis to interrogate putative changes identified by this study.

Objective Systemic lupus erythematosus (SLE) is a systemic autoimmune/inflammatory disease with a strong genetic component. Genetic burden is higher in children when compared to patients with adult-onset SLE, contributing to earlier disease expression and more severe phenotypes. The human leukocyte antigen (HLA) cluster on chromosome 6p21.3 is among the most variable genomic regions, representing a major risk-factor for SLE in adults. Its impact on juvenile-onset (j)SLE remains largely unstudied. Methods High-resolution sequencing of HLA class I (A, B, C), class II (DRB1, DQA1, DQB1) and class III (complement C2) was undertaken in the multi-ancestral UK JSLE Cohort including participants of Caucasian (n=151, 48.8%), Asian (n=108, 35.0%) and African/Caribbean (n=50, 16.2%) descent. Considering ancestral variation, clinical associations were tested at the level of alleles (2-field resolution), associated HLA protein sequences (antigen binding domains, 4-field resolution), and extended haplotypes (DRh). Results Although important ancestral recombination was reported for HLA-DR2 and -DR3 haplotypes, risk associated with jSLE was conserved at related alleles (DR2h: DRB1*15:01, DQA*01:02, DQB1*06:02; DR3h: C*07:02 [Asian], B*08:01, C2 rs9332730 [Asian], DRB1*03:01). HLA-DR7 haplotypes (DRB1*07:01, OR=0.44, 95% CI:0.27-0.72, p=0.0004; DQA1*02:01, OR=0.34, 95% CI:0.21-0.56, p=1.8x10-6) protect Asians from jSLE development. Among 23 clinical variables recorded, the main association was found between low levels of complement C4 in Caucasian carriers of HLA-DR3h. This was not the case in Asians due to recombination with HLA-C*07:02 and integration of the C2 rs9332730 minor allele. Low C4 serum levels associated with HLA-DQA1*01:02 (DR2h) in Caucasians after excluding HLA-DR3h carriers from the analysis. An association between low white blood cell counts and HLA-A*03:01P was observed across ancestries. Conclusion Genetic variation in the HLA cluster associates with organ domain involvement (hematological) and complement levels in jSLE. Lupus-associated HLA haplotypes vary between ancestral groups, underscoring the importance of multi-ancestral approaches to genetic studies in SLE and other autoimmune/inflammatory diseases.

Poxviruses are large DNA viruses with an arsenal of immune-modulatory genes, many of which remain uncharacterized. Proteins with ankyrin repeats are distinct features of poxviruses, although the biological functions of ankyrin proteins are not fully understood. Lumpy skin disease virus (LSDV) encodes five proteins with ankyrin repeats. Here, we reveal the role of LSDV012, an ankyrin protein, in conferring resistance to type I interferon (IFN) in cells. Deletion of LSDV012 from LSDV significantly impacted viral replication in the presence of type I IFN, highlighting the importance of LSDV012 in antagonizing type I IFN responses. Further investigation revealed that LSDV012 interacted with interferon-induced proteins with tetratricopeptide repeats (IFITs), particularly IFIT1, altering its subcellular localization, interacting with its C-terminus and inhibiting its RNA-binding ability without inducing its degradation. Phylogenetic analysis demonstrated that LSDV012 orthologs are conserved in capripoxviruses and cervidpoxviruses, and exhibit host species-specific interactions with IFIT1. Notably, LSDV012 was able to rescue the degradation of IFIT1 mediated by VACV C9. These findings provide novel insights into the viral strategies employed by LSDV to subvert host antiviral defenses and underscore the evolutionary adaptations of poxvirus ankyrin proteins in host species-specific immune evasion.

Liquid-liquid phase separation (LLPS) is a fundamental mechanism for the formation of membrane-less organelles, enabling cells to compartmentalize biochemical processes without membrane boundaries. In viral infections, LLPS is increasingly recognized as a strategy for organizing replication and transcriptional machinery. Here, we report that H5, a DNA-binding protein of vaccinia virus (VACV) could undergo LLPS through its N-terminal intrinsically disordered region (IDR). H5 forms dynamic and reversible condensates in both transfected and vacv infected cells, a property also observed with H5 orthologs from mpox virus and lumpy skin disease virus. Fluorescence recovery after photobleaching (FRAP) assays confirmed the liquid-like behavior of H5 condensates. Using structure-guided mutagenesis and phosphoproteomics, we identified two critical phosphorylation sites within the IDR, S127 and S130, which are essential for the interaction between H5 and DNA. These modifications are mediated redundantly by host proteins and viral B1 kinases. Mutations at these residues inhibit the binding of H5 to DNA, thereby directly or indirectly abolish LLPS formation, and impair viral replication factory assembly, leading to a marked reduction in viral DNA replication and progeny production, without affecting the synthesis of H5 or its subcellular localization. Our findings indicate that these two serine residues of H5 contribute to its interaction with DNA and the formation of LLPS, a process that may help organize viral replication compartments and facilitate interactions with key components of the DNA polymerase complex. This study uncovers a previously uncharacterized mechanism by which the poxvirus H5 protein promotes viral factory assembly and coordinates replication, and identifies a conserved regulatory axis that may serve as a potential therapeutic target across poxvirus species.

SUMMARY Many members of the poxvirus family are important zoonotic pathogens that pose a significant threat to human and animal health worldwide. Autophagy is a multi-step degradation pathway within cells, and one of its primary biological functions includes the clearance of invading viruses. Nevertheless, the interplay between poxviruses and host cell autophagy has not been fully elucidated. Here, we demonstrate that vaccinia virus (VACV) and lumpy skin disease virus (LSDV) induce incomplete autophagy and inhibit the fusion of autophagosomes and lysosomes, while modified vaccinia virus Ankara (MVA), an attenuated strain of VACV unable to replicate in almost all human cells, does not. Additionally, we screened and identified the VACV protein A52 as a key factor that obstruct the formation of autolysosomes. Mechanistically, A52 interacts with SNAP29 and inhibits its interaction with STX17 and VAMP8, both of which are binding partners of SNAP29 and are essential for complete autophagy. Moreover, A52 promotes the proteasomal degradation of SNAP29, which facilitates viral replication. We further revealed that SNAP29 functions as a restriction factor for MVA, as the suppression of SNAP29 allowed the replication of MVA in human cells. In summary, our data present a molecular mechanism by which poxviruses manipulate the cellular autophagic machinery and provide additional explanation for the restriction of MVA in human cells.

In humans, various pathologies have been associated with snoRNAs. Others have shown that the expression of a non-canonical snoRNA Jouvence is involved in lifespan determination in relation to gut homeostasis. As snoRNAs are evolutionary conserved, both structurally and functionally, a jouvence orthologue has been identified in humans. This study aimed to locate the Jouvence snoRNA in the horse genome. Using our previous snoRNA data in ageing equine cartilage along with equine genome data we identified a putative equine Jouvence snoRNA gene. ECABCGRLG0000000730 was the primary candidate horse Jouvence snoRNA. The expression of equine Jouvence snoRNA was increased in ageing equine cartilage. ### Competing Interest Statement The authors have declared no competing interest.

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1β and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

Goatpoxvirus (GTPV), sheeppoxvius (SPPV), and the Lumpy skin disease virus (LSDV) is a Capripoxvirus belonging to the family poxviridae. They can cause significant economic losses in countries where this disease are endemic. However, effective and convenient diagnostic tools against sera antibody are not readily available until now. Toward this goal, a polyclonal antibody competitive enzyme-linked immunosorbent assay (c-ELISA) of detecting serogroup-specific antibody is established based on major LSDV antigen A33. Serum samples (n = 605) were collected to optimize the c-ELISA from different areas. The cut-off value for the c-ELISA was estimate using percent inhibition (PI) values. The diagnostic performance of test including sensitivity (sn) and specificity (sp) were obtained by receiver operator characteristic (ROC) analysis. Among these analysis, > 57.61% PI value was accepted as cut-off of the c-ELISA, the diagnostic sn an diagnostic sp were reached to 96.4% and 98.5%, at > 95% confidence interval. These results show that the developed competitive ELISA is sensitive, specific, and reliable, which make it appropriate for serological investigation.

正痘病毒属病毒的A33和H3L蛋白是其成员共有的中和抗体的两个主要靶标.为检测鼠痘病毒(ectromelia virus,ECTV)A33和H3L蛋白同源物的免疫原性及抗体中和活性,以ECTV-Moscow株基因组DNA为模板,PCR扩增A3 3和H3L同源基因EVM135、EVM085目的序列,分别构建pET30a-EVM135、pET30-EVM085原核表达载体,转化Rosetta感受态细胞,IPTG诱导表达,利用镍层析柱纯化并进行逐步透析复性,免疫兔制备多克隆抗体.建立间接ELISA方法测定其抗体效价分别达1∶120 000和1∶360 000,Western blot和IFA证实该多克隆抗体具有良好的特异性和反应性;ECTV病毒中和试验表明抗EVM135多克隆抗体中和效价为1∶16,而抗EVM085多克隆抗体效价为1∶128.本研究通过高效表达EVM135和EVM085蛋白,并分析其免疫原性及抗体中和活性,为深入研究痘病毒致病和免疫机理,进而快速建立其诊断方法,为猴痘防控技术措施的研究奠定基础.

本研究从疑似爆发伪狂犬病(pseudorabies,PR)猪场死亡猪中分离到1株病毒,经PCR检测、主要毒力基因gB、gC、gD和gE测序及遗传关系分析,发现它与国内流行的PRV变异毒株具有相似的突变特征,氨基酸序列与HN-ZZ/Swine(MH321405)变异株亲缘关系最近,将其鉴定为伪狂犬病变异病毒(pseudorabies virus,PRV),并命名为PRVHN-2017株,发现该毒株在Vero细胞上具有较强的适应性.为进一步探讨PRV HN-2017株的致病性,将该变异株按不同剂量经滴鼻途径接种于45日龄的SPF猪,均先后出现了发热、抽搐、呼吸困难等猪伪狂犬病典型症状,其中高剂量感染组在7 d内全部死亡;RT-PCR检测证实该病毒毒株在体内分布较广,在扁桃体中病毒载量最高,其次是脾脏、肺脏、淋巴结、脑组织、肾脏和肝脏.这表明成功分离到1株PRV变异毒株,并证实该毒株对仔猪具有很强的致病性,能导致多种重要组织器官的不同程度的损害.

Osteoarthritis is the most common degenerative joint disorder. MicroRNAs are gene expression regulators that act post-transcriptionally to control tissue homeostasis. Microarray analysis was undertaken in osteoarthritic intact, lesioned and young intact cartilage. Principal component analysis showed that young intact cartilage samples were clustered together; osteoarthritic samples had a wider distribution; and osteoarthritic intact samples were separated into two subgroups, osteoarthritic-Intact-1 and osteoarthritic-Intact-2. We identified 318 differentially expressed microRNAs between young intact and osteoarthritic lesioned cartilage, 477 between young intact and osteoarthritic-Intact-1 cartilage and 332 between young intact and osteoarthritic-Intact-2 cartilage samples. For a selected list of differentially expressed microRNAs, results were verified in additional cartilage samples using qPCR. Of the validated DE microRNAs, four-miR-107, miR-143-3p, miR-361-5p and miR-379-5p-were selected for further experiments in human primary chondrocytes treated with IL-1β. Expression of these microRNAs decreased in human primary chondrocytes treated with IL-1β. For miR-107 and miR-143-3p, gain- and loss-of-function approaches were undertaken and associated target genes and molecular pathways were investigated using qPCR and mass spectrometry proteomics. Analyses showed that WNT4 and IHH, predicted targets of miR-107, had increased expression in osteoarthritic cartilage compared to young intact cartilage and in primary chondrocytes treated with miR-107 inhibitor, and decreased expression in primary chondrocytes treated with miR-107 mimic, suggesting a role of miR-107 in chondrocyte survival and proliferation. In addition, we identified an association between miR-143-3p and EIF2 signalling and cell survival. Our work supports the role of miR-107 and miR-143-3p in important chondrocyte mechanisms regulating proliferation, hypertrophy and protein translation.

细胞程序性死亡是一种受基因调控的细胞死亡方式,包括细胞凋亡、焦亡、坏死性凋亡、铁死亡、自噬等,这些多元化的细胞程序性死亡方式在病毒的复制和传播中发挥重要作用.其中,痘病毒通过编码大量的病毒蛋白(例如E3L、F1L等)参与介导痘病毒免疫逃逸,抵抗宿主细胞死亡,以此维持自身复制.本文综述了痘病毒E3L蛋白在调控细胞凋亡、焦亡、坏死性凋亡中的作用,深入剖析了 PKR、F1L和Zα结构域介导的E3L相关细胞程序性死亡分子机制,以期为痘病毒免疫逃逸机制探索、抗病毒药物开发等提供新思路.

Introduction: The anterior cruciate ligament (ACL) is susceptible to degeneration, resulting in joint pain, reduced mobility, and osteoarthritis development. There is currently a paucity of knowledge on how anterior cruciate ligament degeneration and disease leads to osteoarthritis. Small non-coding RNAs (sncRNAs), such as microRNAs and small nucleolar RNA (snoRNA), have diverse roles, including regulation of gene expression.Methods: We profiled the sncRNAs of diseased osteoarthritic ACLs to provide novel insights into osteoarthritis development. Small RNA sequencing from the ACLs of non- or end-stage human osteoarthritic knee joints was performed. Significantly differentially expressed sncRNAs were defined, and bioinformatics analysis was undertaken.Results and Discussion: A total of 184 sncRNAs were differentially expressed: 68 small nucleolar RNAs, 26 small nuclear RNAs (snRNAs), and 90 microRNAs. We identified both novel and recognized (miR-206, -365, and -29b and -29c) osteoarthritis-related microRNAs and other sncRNAs (including SNORD72, SNORD113, and SNORD114). Significant pathway enrichment of differentially expressed miRNAs includes differentiation of the muscle, inflammation, proliferation of chondrocytes, and fibrosis. Putative mRNAs of the microRNA target genes were associated with the canonical pathways “hepatic fibrosis signaling” and “osteoarthritis.” The establishing sncRNA signatures of ACL disease during osteoarthritis could serve as novel biomarkers and potential therapeutic targets in ACL degeneration and osteoarthritis development.

The monkeypox epidemic has attracted global attention to poxviruses. The cytoplasmic replication of poxviruses requires extensive protein synthesis, challenging the capacity of the endoplasmic reticulum (ER). However, the role of the ER in the life cycle of poxviruses is unclear. In this study, we demonstrate that infection with the lumpy skin disease virus (LSDV), a member of the poxvirus family, causes ER stress in vivo and in vitro, further facilitating the activation of the unfolded protein response (UPR). Although UPR activation aids in the restoration of the cellular environment, its significance in the LSDV life cycle remains unclear. Furthermore, the significance of ER imbalance for viral replication is also unknown. We show that LSDV replication is hampered by an unbalanced ER environment. In addition, we verify that the LSDV replication depends on the activation of PERK‐eIF2α and IRE1‐XBP1 signaling cascades rather than ATF6, implying that global translation and reduced XBP1 cleavage are deleterious to LSDV replication. Taken together, these findings indicate that LSDV is involved in the repression of global translational signaling, ER chaperone transcription, and ATF6 cleavage from the Golgi into the nucleus, thereby maintaining cell homeostasis; moreover, PERK and IRE1 activation contribute to LSDV replication. Our findings suggest that targeting UPR elements may be applied in response to infection from LSDV or even other poxviruses, such as monkeypox.

Guanylate-binding proteins (GBPs) are highly expressed interferon-stimulated genes (ISGs) that play significant roles in protecting against invading pathogens. Although their functions in response to RNA viruses have been extensively investigated, there is limited information available regarding their role in DNA viruses, particularly poxviruses. Ectromelia virus (ECTV), a member of the orthopoxvirus genus, is a large double-stranded DNA virus closely related to the monkeypox virus and variola virus. It has been intensively studied as a highly effective model virus. According to the study, GBP2 overexpression suppresses ECTV replication in a dose-dependent manner, while GBP2 knockdown promotes ECTV infection. Additionally, it was discovered that GBP2 primarily functions through its N-terminal GTPase activity, and the inhibitory effect of GBP2 was disrupted in the GTP-binding-impaired mutant GBP2K51A. This study is the first to demonstrate the inhibitory effect of GBP2 on ECTV, and it offers insights into innovative antiviral strategies.

目的 克隆羊口疮病毒020基因,构建其重组真核表达质粒并转染HeLa细胞,分析020基因编码E3L蛋白的亚细胞定位,分析该蛋白生物信息学特征.方法 提取羊口疮病毒基因组,PCR扩增020基因并构建重组真核表达质粒,转染HeLa细胞,采用Western blot检测其在该细胞中的表达,免疫荧光法检测该蛋白的亚细胞定位.利用DNAstar软件分析不同毒株ORFV E3L氨基酸序列特点及其遗传演化关系;利用SOMPA软件分析其二级结构;利用SignalP4.0软件预测信号肽;利用TMHMM2.0软件预测跨膜结构;利用NetPhos 3.1预测磷酸化位点;利用NetNGlyc-1.0预测糖基化位点;应用IDEB和SYFPEITHI预测ORFV E3L蛋白的抗原表位.结果 成功克隆得到ORFV 020基因,全长549 bp,编码183个氨基酸;亚细胞定位显示该蛋白定位于细胞核和细胞质.生物信息学分析ORFV E3L蛋白α螺旋约占40.44％、β折叠约占5.46％、延伸链约占16.94％、无规则卷曲约占37.16％;该蛋白无信号肽和跨膜结构,可能存在15个磷酸化位点,7个N-糖基化位点,5个B细胞线性表位,2个CTL表位,3个Th细胞表位,3个B细胞和CTL联合表位,3个B细胞和Th细胞联合表位.结论 ORFV E3L蛋白定位于细胞核和细胞质,含有多个抗原表位,可能具有免疫原性.该蛋白高度保守,具有成为优势保护性抗原的潜力.为进一步揭示ORFV E3L蛋白的功能奠定基础,为ORFV诊断方法的建立及疫苗的研制提供了理论依据.

牛结节性皮肤病(lumpy skin disease,LSD)是由痘病毒科山羊痘病毒属的结节性皮肤病病毒(lumpy skin disease virus,LSDV)感染牛而引起的一种热性、接触性、嗜上皮性病毒病.2019年8月3日,我国首次在新疆维吾尔自治区伊犁州确诊暴发了牛结节性皮肤病疫情,目前LSD已扩散至我国东南、西南、中部及北部等大部分地区且呈蔓延态势.对LSD高效、准确、快速的诊断,是成功控制和根除LSD的基础.本文对LSD的临床特征、病原学、分子生物学和血清学等诊断方法进行综述,为LSD快速有效的诊断及科学防控提供理论支持.

Poxviruses have been associated with humans for centuries. From smallpox to mpox to lumpy skin disease virus (LSDV), members of the poxvirus family have continued to threaten the lives of humans and domestic animals. A complete understanding of poxvirus-mediated cellular processes will aid in the response to challenges from the viruses. In this study, we demonstrate that LSDV infection results in an abnormal ultrastructure of the endoplasmic reticulum (ER) lumen in primary bovine embryonic fibroblast (BEF) cells, and we further show that an ER imbalance occurs in LSDV-infected BEF cells. Additionally, we believe that ER stress-related apoptosis plays a role in the late apoptosis of BEF cells infected with LSDV, primarily through the activation of the CCAAT/enhancer binding protein homologous protein (CHOP)-Caspase-12 signal. In addition to cell apoptosis, a further investigation showed that LSDV could also activate autophagy in BEF cells, providing additional insight into the exact causes of LSDV-induced BEF cell death. Our findings suggest that LSDV-induced BEF cell apoptosis and autophagy may provide new avenues for laboratory diagnosis of lumpy skin disease progression and exploration of BEF cell processes.

To investigate the effect of bovine G3BP1(bG3BP1)on the enzymatic activity of DNA recog-nition receptor cGAS,the total RNA from bovine blood lymphocytes was extracted and reversely tran-scribed into cDNA by RT-PCR and the bG3BP1 gene were cloned.Subsequently,the prokaryotic expression vector pET-28a-SUMO-bG3BP1 was constructed and transformed into competent E.coli cells.The recombi-nant protein bG3BP1 was purified by nickel column affinity chromatography and purified further after removing SUMO label proteins to reach high purity.Finally,the bG3BP1 was used for the enzymatic reac-tion of bovine cGAS(bcGAS)in vitro,and the product of 2'3'-cGAMP was detected by ELISA.The results showed that soluble bG3BP1 protein promoted the enzymatic reaction of bcGAS in vitro and increased the yield of the second messenger molecule 2'3'-cGAMP,which provided technical methods and research ideas for the large-scale production and application of 2'3'-cGAMP.