Mycoplasma bovis (M. bovis) is a globally significant pathogen, causing substantial economic losses to the cattle industry through pneumonia, mastitis, and arthritis. Despite persistent efforts, the development of a consistently effective vaccine remains elusive. This review synthesizes progress across inactivated, attenuated live, and subunit vaccine platforms, highlighting that even promising candidates yield only partial and inconsistent protection. We argue that these shortcomings arise from two principal, intertwined obstacles: the absence of a standardized framework for assessing vaccine efficacy, encompassing unified challenge models, validated immune correlates, and well-defined endpoints, and the pathogen's remarkable ability to subvert diverse host immune defense mechanisms. Consequently, achieving robust protection necessitates a paradigm shift. A deeper mechanistic understanding of host-pathogen interactions, coupled with the strategic application of reverse vaccinology and precise gene-editing technologies, will be essential to design next-generation vaccines that can finally overcome M. bovis immune evasion.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm), is a devastating cattle disease with high morbidity and mortality. Although mass vaccination or stamping-out strategy has controlled CBPP in many countries, it remains a major threat to cattle productivity in sub-Saharan Africa. An attenuated vaccine derived from serial passages of the virulent Ben-1 strain has eradicated CBPP in China, but the underlying immunoprotective mechanisms are unclear. While interferon-inducible GTPases are known to play a crucial role in host defense, IRG-47, an interferon-gamma (IFN-γ)-inducible GTPase, was previously thought to have limited anti-infective activity. Here, we demonstrate that IRG-47 is critical for defending against Mmm and is specifically upregulated by the Ben vaccine strain. Screening potential protective proteins in this vaccine strain revealed that Ben_0691 is a key factor responsible for boosting IRG-47 to combat Mmm infection. Mechanistically, Ben_0691 binds directly to the host cell ATP synthase subunit alpha (ATP5A1) and stabilizes ATP5A1 by reducing its autophagic-degradation. This stabilization promotes IRG-47 expression, which disrupts Mmm's early cell-associativity and provides resistance to Mmm infection. Notably, Ben_0691 N-terminal amino acids 66-72 are required for its interaction with ATP5A1, the induction of IRG-47 and anti-Mmm action. The AMPK-mTOR-IFN-γ pathway is further characterized as an essential signaling for ATP5A1 to facilitate IRG-47 expression. Together, our study reveals a previously unrecognized role of IRG-47 in defending against Mmm infection, and the protective mechanism of Ben_0691 as an IRG-47 activator. These findings provide new insights into vaccine-based protection strategies and may inform future CBPP control strategies.

Mycoplasma bovis is a highly infectious pathogenic microorganism that causes various clinical signs in cattle, including pneumonia, arthritis, and mastitis, often resulting in significant economic losses. The adhesion of M. bovis to host cells is a pivotal step in the infection process, which is a complex process involving multiple pathogenic and host proteins. Molecules involved in M. bovis adhesion and colonization are widely recognized as important virulence factors and often implicated in the infection and pathogenesis. In this study, the Mbov_0510 gene of M. bovis was cloned, and the protein encoded by this gene was expressed and purified. The rabbit polyclonal antibody against this protein was also produced. This protein was shown to be a surface protein and to react with the M. bovis-positive serum. The ability of this protein to adhere to host cells was verified using embryonic bovine lung (EBL) and Madin-Darby bovine kidney (MDBK) cells. Furthermore, the adhesion function of this protein was found to be achieved through interaction with heparin on the host cell surface, and the key region of the protein involved in heparin binding was identified. The conservation of the protein encoded by the Mbov_0510 gene was also analyzed. The results of this study suggest that the protein encoded by the Mbov_0510 gene is a heparin-binding surface membrane protein of M. bovis associated with infection.IMPORTANCEMycoplasmas lack a cell wall, and the membrane proteins interacting with host cells play essential roles in their infection and proliferation processes. In this study, we identified a membrane protein encoded by Mycoplasma bovis that interacts with heparin on the surface of host cells. Heparin is widely distributed in various cells and tissues of the host and serves as a receptor for the infection and invasion of many pathogenic microorganisms. The ability of M. bovis to invade multiple tissues may be related to its heparin-binding capacity. The heparin-binding protein identified in this study is valuable for further research on the infection and invasion mechanisms of M. bovis.

Mycoplasma bovis, a minimal bacterium but a notorious cattle pathogen, leads to serious economic losses. This pathogen binding to host cells is emerging as a complex process involving a broad range of surface-exposed structures. For mycoplasma, adhering to host tissue is the first and crucial step of infection. It is well known that the molecules contributing to microbial cytoadhesion are important virulence factors. Here, we cloned, expressed, and purified the recombinant protein, which is encoded by MBOVJF4278_00820, and induced polyclonal antibodies for it in mice. The cytoadhesive properties of this surface-exposed protein were demonstrated on embryonic bovine lung (EBL) and Madin-Darby bovine kidney (MDBK) cells. Furthermore, heparin as the binding target was confirmed, and the characteristics of the interaction between this protein and heparin have also been analyzed. Our data indicate that the surface-associated MBOVJF4278_00820-encoded protein is a novel adhesion-related factor of Mycoplasma bovis.IMPORTANCEAdhesins are crucial in facilitating Mycoplasma bovis infection. In this study, we identified a specific Mycoplasma bovis adhesin that interacts with heparin on the surface of host cells. Given that heparin is ubiquitously distributed across a wide range of tissue cells, the identification of the heparin-binding adhesin is significant for elucidating how Mycoplasma bovis targets diverse host cells and triggers a spectrum of clinical manifestations.

Mycoplasmas are minimal but notorious bacteria that infect humans and animals. These genome-reduced organisms have evolved strategies to overcome host apoptotic defense and establish persistent infection. Here, using Mycoplasma bovis as a model, we demonstrate that mycoplasma glycine cleavage system (GCS) H protein (GcvH) targets the endoplasmic reticulum (ER) to hijack host apoptosis facilitating bacterial infection. Mechanically, GcvH interacts with the ER-resident kinase Brsk2 and stabilizes it by blocking its autophagic degradation. Brsk2 subsequently disturbs unfolded protein response (UPR) signaling, thereby inhibiting the key apoptotic molecule CHOP expression and ER-mediated intrinsic apoptotic pathway. CHOP mediates a cross-talk between ER- and mitochondria-mediated intrinsic apoptosis. The GcvH N-terminal amino acid 31–35 region is necessary for GcvH interaction with Brsk2, as well as for GcvH to exert anti-apoptotic and potentially pro-infective functions. Notably, targeting Brsk2 to dampen apoptosis may be a conserved strategy for GCS-containing mycoplasmas. Our study reveals a novel role for the conserved metabolic route protein GcvH in Mycoplasma species. It also sheds light on how genome-reduced bacteria exploit a limited number of genomic proteins to resist host cell apoptosis thereby facilitating pathogenesis.

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides ( Mmm ) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study’s protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens. Key points • A Mmm-specific diagnostic marker was obtained based on protein characteristics. • A cELISA was established for CBPP serum antibody detection. • The serological investigation was conducted for CBPP in the border areas of China.

Smallpox is a highly contagious human disease caused by the variola virus. Although the disease was eliminated in 1979 due to its highly contagious nature and historical pathogenicity, with a mortality rate of up to 30%, this virus is an important candidate for biological weapons. Currently, vaccines are the critical measures to prevent this virus infection and spread. In this study, we designed a peptide vaccine using immunoinformatics tools, which have the potential to activate human immunity against variola virus infection efficiently. The design of peptides derives from vaccine-candidate proteins showing protective potential in vaccinia WR strains. Potential non-toxic and nonallergenic T-cell and B-cell binding and cytokine-inducing epitopes were then screened through a priority prediction using special linkers to connect B-cell epitopes and T-cell epitopes, and an appropriate adjuvant was added to the vaccine construction to enhance the immunogenicity of the peptide vaccine. The 3D structure display, docking, and free energy calculation analysis indicate that the binding affinity between the vaccine peptide and Toll-like receptor 3 is high, and the vaccine receptor complex is highly stable. Notably, the vaccine we designed is obtained from the protective protein of the vaccinia and combined with preventive measures to avoid side effects. This vaccine is highly likely to produce an effective and safe immune response against the variola virus infection in the body. IMPORTANCE:In this work, we designed a vaccine with a cluster of multiple T-cell/B-cell epitopes, which should be effective in inducing systematic immune responses against variola virus infection. Besides, this work also provides a reference in vaccine design for preventing monkeypox virus infection, which is currently prevalent.

为探究牛支原体(M.bovis)脂质相关膜蛋白(LAMPs)抑制宿主细胞凋亡的分子机制,本研究利用实验室前期经AKTA分子筛分成5组的M.bovis TJ株LAMPs分别刺激胎牛肺(EBL)细胞,经western blot检测各组LAMPs对EBL细胞中凋亡执行分子Caspase-3剪切激活水平的影响,经CCK-8试验检测筛选出的目标LAMPs对EBL细胞活力的影响,并对其进行蛋白质谱测序分析.结果显示,D组LAMPs能够抑制Caspase-3的剪切激活水平,并可以极显著提高EBL细胞活力,结合蛋白质谱测序结果以及生物信息学软件预测蛋白功能,选定VSPHB0801-4、TPP、DJ-1、EF-TU、GAPDH以及GLCP为候选蛋白进行后续试验.经PCR扩增dj-1基因,重叠延伸PCR扩增ef-tu和glcp融合基因,vsphb0801-4、tpp和gapdh基因片段则由华大基因公司合成,将上述基因片段分别克隆至相应的原核表达载体中,构建重组质粒pET-28a-vsphb0801-4、pET-28a-tpp、pET-32a-dj-1、pET-32a-ef-tu、pEGX-6P-1-gapdh和pMAL-c5X-glcp,并分别转化大肠杆菌BL21(DE3),经IPTG诱导后通过亲和层析纯化目的蛋白,SDS-PAGE检测重组蛋白的纯化效果.结果显示,获得了纯度较高的6个重组蛋白.将不同浓度的6个重组蛋白分别刺激EBL细胞,经western blot验证重组蛋白是否具有抑制EBL细胞凋亡的作用,结果显示,只有重组蛋白GLCP能够抑制EBL细胞凋亡.因此,将其作为靶标蛋白进一步探究其抑制EBL细胞凋亡的分子机制.将不同浓度的重组蛋白GLCP分别刺激EBL细胞后,经western blot检测经典凋亡通路介导分子Caspase-8、Caspase-9和Caspase-12的剪切激活水平、激光共聚焦显微镜检测EBL细胞线粒体膜电位,以及western blot检测线粒体介导的凋亡通路关键分子Bcl-2、Bax、CytC、Apaf-1以及剪切的多聚ADP核糖聚合酶-1(PARP1)蛋白的表达水平.结果显示,重组蛋白GLCP主要抑制Caspase-9的剪切激活水平,提高线粒体膜电位,并上调线粒体介导的凋亡通路关键分子Bcl-2蛋白的表达水平,以及下调Bax、CytC、Apaf-1和剪切的PARP1蛋白表达水平.以上实验结果表明,重组蛋白GLCP主要通过线粒体介导的凋亡通路抑制EBL细胞凋亡.本研究首次筛选到抑制EBL细胞凋亡的M.bovis脂质相关膜蛋白GLCP,并初步解析了其抑制宿主细胞凋亡的分子机制,为M.bovis持续性感染机制的研究提供参考依据.

The unfolded protein response (UPR) plays a crucial role in Mycoplasma hyopneumoniae (M. hyopneumoniae) pathogenesis. We previously demonstrated that M. hyopneumoniae interferes with the host UPR to foster bacterial adhesion and infection. However, the underlying molecular mechanism of this UPR modulation is unclear. Here, we report that M. hyopneumoniae membrane protein Mhp271 interacts with host GRP78, a master regulator of UPR localized to the porcine tracheal epithelial cells (PTECs) surface. The interaction of Mhp271 with GRP78 reduces the porcine beta-defensin 2 (PBD-2) production, thereby facilitating M. hyopneumoniae adherence and infection. Furthermore, the R1-2 repeat region of Mhp271 is crucial for GRP78 binding and the regulation of PBD-2 expression. Intriguingly, a coimmunoprecipitation (Co-IP) assay and molecular docking prediction indicated that the ATP, rather than the substrate-binding domain of GRP78, is targeted by Mhp271 R1-2. Overall, our findings identify host GRP78 as a target for M. hyopneumoniae Mhp271 modulating the host UPR to facilitate M. hyopneumoniae adherence and infection.

旨在制备抗丝状支原体丝状亚种(Mmm)的特异性单克隆抗体(MAb),为牛传染性胸膜肺炎(CBPP)病原诊断的免疫学方法提供特异性抗体.本研究利用生物信息学技术分析了 Mmm国内分离株Ben-1不同传代株的全基因组序列,选取M0071蛋白作为研究对象.将原核表达的可溶性重组蛋白M0071(rM0071)作为免疫原免疫BALB/c小鼠,通过有限稀释法和间接ELISA方法筛选得到能稳定分泌抗rM0071蛋白的单克隆抗体的杂交瘤细胞株.进一步制备单抗腹水并纯化,利用Western blot方法对该单抗进行特异性鉴定,同时测定其抗体效价和抗体亚类.随后利用间接免疫荧光试验(IFA)评价该单抗对细胞感染Mmm的检测能力.结果表明成功获得1株单克隆细胞株3C4A1,将其分泌抗体命名为MAb 3C4A1.特异性结果表明,MAb 3C4A1能与Mmm的分离株和标准株发生特异性反应,而不与山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、牛鼻支原体、无乳支原体、牛支原体、leachii支原体和牛A型巴氏杆菌等发生反应.抗体亚类鉴定MAb 3C4A1属于IgG1亚类、轻链为κ链.经间接ELISA测定其抗体效价为1:256 000.IFA试验结果表明,MAb 3C4A1仅与感染EBL细胞的Mmm发生绿色荧光反应,而与牛鼻支原体、无乳支原体、牛支原体感染的细胞不发生荧光反应,特异性良好.本研究制备的MAb 3C4A1具有良好的特异性和免疫反应性,可作为CBPP病原免疫学诊断的工具,为进一步研制CBPP病原鉴别诊断试剂盒提供了基础材料.

为筛选并鉴定免疫反应原性良好的牛支原体(M.bovis)蛋白,本研究提取M.bovis TJ株脂质相关膜蛋白(LAMPs),通过AKTA分子筛筛选,将M.bovis LAMPs分为5组,经dot blot试验鉴定各组LAMPs的免疫反应原性,并经蛋白质谱分析其蛋白组分.结果显示,5组LAMPs中有1组膜蛋白反应原性较好,且结合蛋白匹配度得分的排名顺序和基因注释,选定P48、DnaK和L-LDH为候选蛋白.将该3种蛋白经原核表达、纯化及western blot检测其反应原性;并进一步分别以该3种重组蛋白为包被抗原,采用ELISA方法(rP48-ELISA、rDnaK-ELISA、rL-LDH-ELISA)检测并利用Graphpad prism 8软件分析并计算该3种方法的特异性、敏感性及其与牛鼻支原体、丝状支原体丝状亚种SC型、牛病毒性腹泻病毒、口蹄疫病毒、牛传染性鼻气管炎病毒阳性血清的交叉反应性.Western blot结果显示,3种重组蛋白均能与M.bovis阳性血清反应,反应原性较好;ELISA结果显示,rP48-ELISA、rDnaK-ELISA、rL-LDH-ELISA方法的特异性分别为96％、94％和98％,敏感性分别为98％、92％和94％.交叉反应性试验结果显示,M.bovis rDnaK和rL-LDH均能与牛鼻支原体阳性血清发生交叉反应,而M.bovis rP48与牛其他临床常见呼吸道病原阳性血清均无交叉反应,表明其具有作为检测M.bovis ELISA方法候选包被抗原的潜力.本研究为进一步筛选M.bovis亚单位疫苗抗原及快速诊断方法的候选抗原奠定实验基础.

Bluetongue (BT) is an arbovirus-borne disease of ruminants caused by bluetongue virus (BTV) that has the potential to have a serious economic impact. Currently available commercial vaccines include attenuated vaccines and inactivated vaccines, both of which have achieved great success in the prevention and control of BTV. However, these vaccines cannot distinguish between infected animals and immunized animals. To control outbreaks of BTV, the development of labeled vaccines is urgently needed. In this study, we used the plasmid-based reverse genetics system (RGS) of BTV to rescue four recombinant viruses in which HA (influenza hemagglutinin) tags were inserted at different sites of VP2. In vitro, the recombinant tagged viruses exhibited morphologies, plaque, and growth kinetics similar to the parental BTV-16, and expressed both VP2 and HA tag. Subsequently, the selected recombinant tagged viruses were prepared as inactivated vaccines to immunize IFNAR(-/-) mice and sheep, and serological detection results of anti-HA antibody provided discriminative detection. In summary, we used plasmid-based RGS to rescue BTV recombinant viruses with HA tags inserted into VP2, and detected several sites on VP2 that can accommodate HA tags. Some of the recombinant tagged viruses have potential to be developed into distinctive inactivated vaccines.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

Background Bluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription. Result In this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others. Conclusion In this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.

为建立蓝舌病病毒(BTV) 10质粒反向遗传操作系统,本研究将BTV 16型BN96/16株10个基因节段分别克隆至pS111载体相应的酶切位点,构建10个重组质粒转染BSR细胞,拯救获得重组病毒,命名为rBTV-16.测序结果显示,拯救病毒rBTV-16与其亲本病毒wtBTV-16的核苷酸序列完全相同;经电镜观察,可见典型形态的环状病毒属病毒粒子;间接免疫荧光试验结果显示,rBTV-16能够感染细胞;病毒基因组检测结果显示,rBTV-16各个基因节段的长度与亲本病毒完全一致;病毒生长曲线的测定结果显示,rBTV-16与亲本病毒具有一致的复制特性.本研究建立的10质粒系统比传统体外转录拯救系统的操作过程简便,提高了拯救病毒的效率,为进一步深入研究BTV的致病机理奠定了基础.

Bluetongue virus (BTV), a complex double-stranded segmented RNA virus, has been found to initiate cellular autophagy for its own benefit. Here, with a view to understanding the underlying mechanisms, we first systematically dissected the exact signaling network in BTV-induced autophagy. We found that the activity of mTOR, a crucial pivot, was inhibited by BTV1 infection, subsequently leading to downstream p70S6K suppression and autophagy initiation. We then explored the upstream regulators of mTOR and analyzed their activities via a series of assays. We found BTV1-induced autophagy to be independent of the ERK1/2 signaling pathway. However, the BTV1-induced inhibition of PI3K/Akt was found to be partially responsible for mTOR inactivation and subsequent autophagy initiation. Furthermore, we found unexpectedly that AMPK seemed to play a more important role in BTV1-induced autophagy. Elevated [Ca 2+ ] cyto -mediated activation of CaMKKβ exactly managed the activation of AMPK, which then positively regulated autophagy through suppressing mTOR. We must emphasize that TSC2 is a fatal mediator between upstream Akt or AMPK and downstream mTOR through its phosphorylation. Taken together, our data suggested that the BTV1-induced inhibition of the Akt-TSC2-mTOR pathway and the upregulation of the AMPK-TSC2-mTOR pathway both contributed to autophagy initiation and further favored virus replication.

Bluetongue virus (BTV) has been found to trigger autophagy to favor its replication, but the underlying mechanisms have not been clarified. Here, we show that cellular energy metabolism is involved in BTV-induced autophagy. Cellular ATP synthesis was impaired by BTV1 infection, causing metabolic stress, which was responsible for activation of autophagy, since the conversion of LC3 and aggregation of GFP-LC3 (autophagy markers) were suppressed when infection-caused energy depletion was reversed via MP (metabolic substrate) treatment. The reduced virus yields with MP further supported this view. Overall, our findings suggest that BTV1-induced disruption of cellular energy metabolism contributes to autophagy, and this provides new insights into BTV-host interactions.

蓝舌病病毒(bluetongue virus,BTV)编码4种非结构蛋白(NS1～NS4),其中NS4是新发现的非结构蛋白.为深入探索该新型非结构蛋白的功能,本研究首先采用生物信息学分析其序列结构特征及可能的功能域,结果发现NS4蛋白的N端α-螺旋结构域中具有富含碱性氨基酸的核定位信号(NLS),而C端α-螺旋结构域中具有亮氨酸拉链结构的核输出信号(NES).为进一步证实NS4的亚细胞定位,本试验构建了表达NS4全长及其缺失突变体的重组质粒,结果显示缺失NLS的NS4重组蛋白弥散在胞浆,而缺失NES后NS4重组蛋白只定位于细胞核,表明NS4蛋白亚细胞定位具有核—胞浆穿梭过程.最后结合3D同源建模发现NS4与CCAAT增强子结合蛋白(C/EBPβ)等具有较高的同源性,据此推测NS4可能为转录调控蛋白,与DNA或其他转录因子存在相互作用.总之,本研究为NS4蛋白的功能研究提供了更多新线索和新思路,填补了BTV基础研究的空白.

Bluetongue (BT), which is caused by the BT virus (BTV), is an important disease in ruminants that leads to significant economic losses in the husbandry industry. To detect BTV-specific antibodies in serum, a protein chip detection method based on a novel solid supporting material known as polymer-coated initiator-integrated poly (dimethyl siloxane) (iPDMS) was developed. With a threshold of 25% (signal-to-noise percentage), the sensitivity and specificity of the protein chip were 98.6% and 94.8%, respectively. Furthermore, spot serum samples obtained from six provinces of China were tested with the protein chip and a commercially available BTV enzyme-linked immunosorbent assay (ELISA) kit (IDEXX). Of 615 samples, BTV-specific antibodies were detected in 200 (32.52%) by the protein chip and in 176 (28.62%) by the IDEXX BTV ELISA kit. Comparison of the protein chip with the commercial IDEXX BTV ELISA kit yielded the following spot serum detection results: a total coincidence, a negative coincidence and a positive coincidence of 95.12%, 99.28% and 86.5%, respectively. With the protein chip, the BTV-specific serum antibody was detected in samples from all six provinces, and the positive rates ranged from 4.12 to 74.4%. These results indicate that this protein chip detection method based on iPDMS is useful for the serological diagnosis of BTV infection and for epidemiological investigation.

蓝舌病病毒(bluetongue virus,BTV)的结构主要由3层衣壳蛋白组成,其中VP2、VP5蛋白构成了BTV的外层衣壳,VP7蛋白构成了BTV的中间衣壳,最内层衣壳则由VP3蛋白构成.VP2、VP5及VP7蛋白在BTV侵染宿主细胞的过程中起着非常重要的作用.为了研究BTV与宿主细胞相互作用的分子机制,本研究将BTV的VP2、VP5、VP7基因分别克隆到pGBKT7载体中,成功构建了pGBKT7-VP2、pGBKT7-VP5与pGBKT7-VP7 3个诱饵质粒,且通过自激活和毒性验证,证明所构建的3个质粒均无自激活作用,对酵母细胞无毒性作用.本研究为今后利用酵母双杂交筛选VP2、VP5、VP7蛋白中与宿主细胞相互作用的蛋白做好了铺垫,为深入研究BTV与宿主细胞的相互作用奠定了基础.