为研制鸡异质核糖核蛋白AB(hnRNPAB)单克隆抗体,为其生物学功能研究提供关键试剂,构建重组质粒pET-30a-hnRNPAB,转化感受态细胞E.coli BL21,经IPTG诱导表达,并通过树脂Ni对可溶性表达的鸡hnRNPAB蛋白进行亲和层析纯化之后免疫Balb/c小鼠,利用细胞融合技术制备抗鸡hnRNPAB的单克隆抗体细胞株,最终通过间接酶联免疫吸附试验(iELISA)筛选获得1株单克隆抗体杂交瘤细胞株5H2-H1.间接免疫荧光试验(IFA)和蛋白质免疫印迹(Western blot)分析表明,5H5-H1单克隆抗体可特异性识别鸡胚成纤维细胞(CEF)表达的hnRNPAB蛋白.同时IFA和间接免疫细胞化学试验(ICA)结果还显示,除了特异性识别鸡hnRNPAB蛋白之外,5H5-H1单克隆抗体还可以与源自人、鼠、猴、牛和猪在内的5种常见哺乳动物细胞系表达的hnRNPAB发生特异性反应.

Zeranol (α-zearalanol) has been used as a growth promoter in livestock since 1969 in some non-EU countries; the residues of zeranol and its five analogues in animal origin foods may endanger human health due to their strong estrogenic and anabolic activities. Therefore, it is urgent to establish simple, rapid, real-time, broad-spectrum and high-sensitivity detection methods for the residues of zeranol and its analogues. In this study, an ultrasensitive indirect-competition enzyme-linked immunosorbent assay (ic-ELISA) was established for the rapid multi-residue detection of zeranol and its five analogues in cattle origin samples, which was based on a broad-spectrum monoclonal antibody (mAb) that specifically bound to zeranol and its analogues with high sensitivity. The half maximal inhibitory concentration (IC50) values for zeranol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol, and zearalenone were 0.103, 0.080, 0.161, 0.177, 0.254, and 0.194 ng mL-1, respectively, the recovery rates of cattle origin samples spiked with zeranol ranged from 79.2-104.2%, and the coefficient of variation (CV) values were less than 11.4%. Excellent correlation (R2 = 0.9845) was obtained between the results of HPLC-MS/MS and ic-ELISA. In conclusion, the developed ic-ELISA could be employed as an ultrasensitive and broad-spectrum detection method for monitoring trace ZEN residues in cattle origin foods.

Gaeumannomyces graminis var. tritici is a soil borne pathogenic fungus associated with wheat roots. The accurate quantification of gene expression during the process of infection might be helpful to understand the pathogenic molecular mechanism. However, this method requires suitable reference genes for transcript normalization. In this study, nine candidate reference genes were chosen, and the specificity of the primers were investigated by melting curves of PCR products. The expression stability of these nine candidates was determined with three programs-geNorm, Norm Finder, and Best Keeper. TUBβ was identified as the most stable reference gene. Furthermore, the exopolygalacturonase gene (ExoPG) was selected to verify the reliability of TUBβ expression. The expression profile of ExoPG assessed using TUBβ agreed with the results of digital gene expression analysis by RNA-Seq. This study is the first systematic exploration of the optimal reference genes in the infection process of Gaeumannomyces graminis var. tritici.

为了得到一种能够高通量检测细胞培养物的方法,并将其用于筛选特异性强、敏感度高的抗猪繁殖与呼吸综合征病毒(PRRSV)单克隆抗体.用每孔能感染约100个细胞的病毒量接种单层覆盖96孔板的Marc-145细胞,12 h后用含3％H2 O2的甲醇固定细胞,以制备免疫过氧化物酶单层细胞试验(IPMA)反应板.以100μL/孔的量将融合后继续培养10 d的杂交瘤细胞的培养上清加入至IPMA反应板,以辣根过氧化物酶标记的羊抗鼠IgG-HRP作为二抗,以3-氨基-9-乙基咔唑(AEC)作为显色底物,于倒置显微镜下进行观察.结果表明,共筛选出1D1、7G8等39份PRRSV单克隆抗体,这39份单抗能够使PRRSV中高致病毒株HN07-1和经典毒株BJ-4感染的Marc-145细胞被特异性染色,而对猪瘟病毒、猪伪狂犬病毒、猪流行性腹泻病毒感染的Marc-145细胞无交叉染色.因此,构建的IPMA方法能够敏感、准确地捕捉到PRRSV单克隆抗体.

为了制备猪流感病毒(SIV)核蛋白(NP)的单克隆抗体(MAb),采用差速离心法纯化H1N1和H3 N2亚型SIV后交叉免疫BALB/c小鼠,利用淋巴细胞杂交瘤技术制备杂交瘤细胞,通过建立的免疫过氧化物酶单层细胞试验(IPMA)单克隆抗体检测方法进行筛选.获得3株能稳定分泌抗NP蛋白的杂交瘤细胞,分别命名为16 D5、18 G8和20 C4,将它们诱导小鼠产生的腹水IPMA效价分别为1×10-6、1×10-6和1×10-5.亚型鉴定结果显示,3株单克隆抗体的重链均为IgG1,轻链为κ链.3株单克隆抗体特异识别H1 N1、H3 N2、H5 N1、H7 N9和H9 N2亚型流感病毒,并且与猪圆环病毒2型、猪伪狂犬病毒和猪细小病毒无交叉反应.Western blot检测结果表明,3株杂交瘤细胞培养上清均在56 ku附近出现一条特异性的蛋白质条带,说明针对SIV的NP蛋白制备了3株单克隆抗体.综上,成功制备了针对SIV NP蛋白的3株单克隆抗体,可识别不同亚型的SIV.

为制备及鉴定猪繁殖与呼吸综合征病毒(PRRSV)主要结构糖蛋白GP5单克隆抗体,本研究首先利用切向流系统和琼脂糖凝胶层析对PRRSV高致病性毒株HN07-1进行纯化;将纯化后的病毒作为免疫原免疫小鼠;取免疫后的小鼠脾细胞和骨髓瘤细胞进行融合;通过免疫过氧化物酶单层细胞试验对杂交瘤细胞株进行检测及筛选;利用重组GP5蛋白对阳性单克隆上清进行免疫印迹分析鉴定;经激光扫描共聚焦显微术和流式细胞术进一步鉴定单抗与病毒的结合特性.本研究共筛选到4株针对GP5蛋白的单克隆抗体,分别被命名为2D1G11、2D1C1、4H9D1和8B9D4;通过激光共聚焦显微镜观察发现,以上这4株GP5单抗均可以与病毒特异性结合;进一步通过流式细胞术检测发现,这4株GP5单抗与病毒的结合能力超过N单抗.通过亚型鉴定发现,2D1G11、2D1C1属于IgG2亚型,4H9D1、8B9D4为IgG1亚型.PRRSV GP5蛋白特异性单抗的成功制备,为PRRSV快速诊断试剂研制及免疫识别研究提供了分子手段和工具支持.

由假禾谷镰刀菌Fusarium pseudograminearum引起的小麦茎基腐病是一种重要的土传病害,生产上亟需一种安全有效的防控方法。本研究利用枯草芽胞杆菌YB-05与申嗪霉素复配,并评价该复配制剂对小麦茎基腐病的防治效果。结果显示当申嗪霉素浓度低于500μg/m L时,对YB-05的菌落生长无显著影响;室内盆栽及田间试验表明枯草芽胞杆菌YB-05菌液和1%申嗪霉素悬浮剂按照19:1（vt/vt）复配生物制剂20m L/kg拌种处理发病最轻,均超过了高剂量的单剂处理（枯草芽胞杆菌YB-0530m L/kg拌种处理和1%申嗪霉素2 mL/kg拌种处理）和4.8%适麦丹水悬浮剂2 mL/kg拌种处理,并且具有一定的促生和增产作用。其中,复配制剂室内防治效果为69.8%,在两次的田间调查中防治效果分别为57.8%和45.7%。由此可见,枯草芽胞杆菌与申嗪霉素复配具有协同增效的作用,可以减少化学农药的使用,具有较好的开发利用潜力。

The authors describe an ultrasensitive method for simultaneous detection of neomycin (NEO) and quinolones antibiotics (QNS). It is based on the use of (a) two immuno-nanoprobes (a probe for NEO and a probe for QNS), (b) surface-enhanced Raman scattering (SERS) detection, and (c), a portable lateral flow assay (LFA). The two probes consist of gold nanoparticles (AuNPs) conjugated to the Raman active molecule 4-aminothiophenol (PATP), and to monoclonal antibody against NEO (NEO mAb) or against NOR (NOR mAb). Quantitative detection of NEO and QNS was realized via SERS of the PATP-coated AuNPs captured in the test line of a LFA. Under optimized condition, the visual limits of LFA are 10 ng·mL−1 for NEO and 200 ng·mL−1 for NOR, and with LODs down to 0.37 pg·mL−1 and 0.55 pg·mL−1 by using SERS. The NEO test line is not interfered by the NEO analogues gentamycin, streptomycin and tobramycin, but the NOR test line suffers from different degrees of cross-reactivity (CR) to 12 common other QNS, the CRs ranging from 1.5% to 136%. The recoveries of NEO and NOR from spiked milk samples ranged between 86% and 121%, with relative standard deviations (RSD) from 3% to 6%. The method is highly sensitive, accurate and effective. It may be applied to simultaneous detection of NEO and 8 QNS, including NOR, enoxacin, ciprofloxacin, ofloxacin, fleroxacin, marbofloxacin, enrofloxacin, and pefloxacin.

Porcine epidemic diarrhea virus (PEDV) is a devastating swine infectious disease. Development of high-performance methods to diagnose and evaluate viral immune status remains very important to control PEDV. Here, a recombinant S1 protein-based indirect enzyme-linked immunosorbent assay (rpS1-iELISA) was developed to monitor IgA antibody in the colostrum. Optimized cut-off value of the rpS1-iELISA was determined as 0.448. Results yielded a sensitivity of 96.87% and a specificity of 100.0%. Repeatability tests indicated that the coefficients of variation of the colostrum samples within and between runs were both less than 8%.Test results of 523 field colostrum samples showed that the rpS1-IELISA had excellent agreement with immunofluorescence assay (kappa = 0.958) and better test performance than a commercial ELISA kit. This test will aid in future diagnostics and assessment of the protective levels of mucosal immune response against PEDV by measuring IgA levels in the colostrum.

ABSTRACT Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus. IMPORTANCE PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides structural information on the CD163 SRCR domain for the first time but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.

查尔酮合成酶(chalcone synthase,CHS)是植物抗病抗逆过程中的一个关键酶.根据小麦抑制消减杂交中分离得到的小麦查尔酮合成酶基因片段,结合TAIL-PCR技术从小麦(Triticum aestivum L.)叶片中克隆得到查尔酮合成酶基因,命名为TaCHS,其序列全长为2 543 bp,包含1 264 bp的启动子区、1 185 bp编码区和一个94 bp的内含子.分析显示该基因编码的氨基酸具有CHS家族的所有保守功能位点.同源性分析表明,TaCHS与已报道的其他禾本科植物CHS基因编码的氨基酸序列同源性高达88％以上.启动子序列分析显示TaCHS启动子区域具有光反应元件、植物激素响应元件、真菌诱导元件、MYB结合位点、TATA-Box和CAAT-Box等多种顺式作用元件.TaCHS基因在全蚀菌侵染小麦后的表达开始上调,侵染后4d达到最大值,之后开始下调.以上结果表明TaCHS基因可能与小麦防御全蚀菌侵染有关.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.

A rapid lateral flow colloidal gold immunoassay strip was established in a sandwich format with the colloidal gold-labelled mouse anti-β-conglycinin monoclonal antibody (mAb) and the rabbit anti-β-conglycinin polyclonal antibody (pAb) in order to specifically identify a soybean allergen, β-conglycinin. Soybean and soy derivatives have become ubiquitous in many vegetarian and meat-based food products, and as a result, dietary avoidance has become difficult. Therefore, soybean avoidance is challenging for individuals with soybean allergy. In our study, the selected mAb showed high affinity to the target and no cross-reactivity with other soybean allergens. Based on the sandwich immunoassay principle, the rabbit pAb was applied in the test line with high titre. The assay could be accomplished within 10 min without sophisticated procedures. The limit of detection of the test strip was calculated at 1.66 mg/kg in powdered milk using an optical density scanner that measures relative optical density. The assay showed high specificity for β-conglycinin, with no cross-reactions with other food allergens. The efficacy of the lateral flow test strip to detect β-conglycinin in powdered milk samples ranged between 80.8 and 89.2% with relative standard deviations of less than 7.2%. Therefore, the test strip is useful as a rapid and reliable detection method for β-conglycinin in powdered milk. In addition, this assay is suitable for monitoring β-conglycinin in other processed foods when samples are appropriately extracted.

ABSTRACTMicroRNAs (miRNAs) play an important role in the regulation of immune responses. Previous studies have indicated that dysregulating the miRNAs leads to the immunosuppression of porcine reproductive and respiratory syndrome virus (PRRSV). However, it is not clear how PRRSV regulates the expression of host miRNA, which may lead to immune escape or promote the replication of the virus. The present work suggests that PRRSV upregulated the expression of miR-373 through elevating the expression of specificity protein 1 (Sp1) in MARC-145 cells. Furthermore, this work demonstrated that miR-373 promoted the replication of PRRSV, since miR-373 was a novel negative miRNA for the production of beta interferon (IFN-β) by targeting nuclear factor IA (NFIA), NFIB, interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, and interferon regulatory factor 1 (IRF1). We also found that both NFIA and NFIB were novel proteins for inducing the production of IFN-β, and both of them could inhibit the replication of PRRSV. In conclusion, PRRSV upregulated the expression of miR-373 by elevating the expression of Sp1 and hijacked the host miR-373 to promote the replication of PRRSV by negatively regulating the production of IFN-β.IMPORTANCEPRRSV causes one of the most economically devastating diseases of swine, and there is no effective method for controlling PRRSV. It is not clear how PRRSV inhibits the host's immune response and induces persistent infection. Previous studies have shown that PRRSV inhibited the production of type I IFN, and the treatment of type I IFN could efficiently inhibit the replication of PRRSV, so it will be helpful to design new methods of controlling PRRSV by understanding the molecular mechanism by which PRRSV modulated the production of IFN. The current work shows that miR-373, upregulated by PRRSV, promotes PRRSV replication, since miR-373 impaired the production of IFN-β by targeting NFIA, NFIB, IRAK1, IRAK4, and IRF1, and both NFIA and NFIB were antiviral proteins to PRRSV. In conclusion, this paper revealed a novel mechanism of PRRSV that impaired the production of type I IFN by upregulating miR-373 expression in MARC-145 cells.

In order to investigate the mechanism of porcine endometrial endothelial cells infected by porcine reproductive and respiratory syndrome virus,the effects of CD163 on infection of porcine endometrial endothelial cells by porcine reproductive and respiratory syndrome virus( PRRSV) were studied. The recombinant CD163 plasmid was transfected into porcine endometrial endothelial cells and transgenic cell line was generated. Transcription of CD163 in transgenic cell line was detected by RT-PCR and immuonfluorescence. Transgenic cell line was infected by 0. 1 MOI PRRSV( VR2332 srain). The virus was harvested at different time points post-infection and titrated by Marc-145 cells. The result showed that there was no significant difference between CD163 transgenic cells and vector transgenic cells. In conclusion,CD163 had no significant effection on infection of porcine endometrial endothelial cells by PRRSV.

Balb/c mice were immunized by the Newcastle disease virus (NDV) reference strain F48E8 purified by differential centrifugation,and used for monoclonal antibodies(mAbs) production using hybridoma technology.After screened by a heterologous immunoperoxidase monolayer assay(IPMA) based on the NDV-infected BHK-21 cells,fourteen hybridoma cell lines secreting mAbs specific for NDV were obtained,whose antibody titers of ascites were determined to be 0.50 × 10-2-2.56 × 10-5 to F48E8 and to LaSotain by IPMA respectively.In hemagglutination inhibition(HI) assay,mAbs 1 G6,2C1,4D2,5F2 and 13A5 showed hemagglutination-inhibitory activity with the HI titers of 6-12log2(F48E8) and 9-111og2 (LaSota),of which mAbs 5F2 and 13 A5 showed significant neutralizing activity to both NDV virulent and vaccine strains in the virus neutralization (VN) test with the titers of 1∶400-1∶800 and 1∶25 respective-ly.Furthermore,sandwich ELISA using the recombinant proteins showed that mAb 5F2 reacted with HN protein of NDV,while mAb 13A5 recognized the F protein.In conclusion,the NDV mAbs of 5F2 and 13A5 with neutralization activity were obtained successfully.

Take-all is one of the most destructive root diseases of wheat, however, effective biological control agents (BCAs) for controlling the causal organism, Gaeumannomyces graminis var. tritici (Ggt), are currently unavailable. In this study, 155 putative Bacillus strains isolated from soil were tested as potential BCAs against Ggt. The most effective was strain YB-57, which was identified as B. subtilis using 16S rDNA, morphology and physiology. Strain YB-57 showed as high as 60.9% growth inhibition of Ggt and also exhibited significantly inhibitory activity against seven other fungal phytopathogens, indicating its broad-spectrum antagonism to fungi. Greenhouse studies showed that YB-57 lowered the incidence of the take-all disease by 56.5%, which was similar to that of the fungicide, silthiofam. The antimicrobial activity of crude extracts from strain YB-57 fermentation liquor was relatively stable under a range of temperature, pH and UV light exposures and was also resistant to protease K digestion. The extract was purified, and five types of lipopeptides, iturin isomers, surfactin isomers, plipastatin isomers, bacillomycin isomers and difficidin, were identified by HPLC/ESI-MS/MS. These results suggested that B. subtilis strain YB-57 could be a useful BCA for take-all management.

With the continuous progress on affinity peptide research, it has become more and more popular in pharmacology and medicine. lt is promising to study these viruses affinity peptide to treat infectious diseases. And the analysis on the virus affinity peptide with high selectivity and high sensitivity could provide valuable means for disease detection, treatment as wel as the study on the molecular mechanism of virus affinity peptide. Therefore, we reviewed the bioinformatics pre-diction technologies of computer simulation, molecular docking and homology model-ing, as wel as the research method on analyzing and screening virus affinity pep-tide, such as Phage display technology.

Porcine epidemic diarrhea virus (PEDV) has caused devastating impact on pig-rearing industry in China and current vaccine is not effective against the circulating PEDV variants. In the present study, the full-length genome sequence from a PEDV isolate (CH/HNQX-3/14) was determined. The complete genome sequence analysis showed that the CH/HNQX-3/14 possessed unique deletion regions in the S and ORF3 genes. It was identified as a recombinant strain using phylogenetic analysis and recombination detection program. Further analyses of the full-length sequence suggest that CH/HNQX-3/14 is a natural recombinant between the attenuated vaccine strains (CV777 and DR13) and circulating wild-type strain (CH/ZMDZY/11). The recombination occurred not only in structural protein-coding region (S1 and N genes) but also in non-structural protein-coding region (replicases 1a and ORF3 genes). These results provided new evidence that PEDV strains circulating in China underwent recombination between vaccine and field strains, suggesting that recombination contributes to the genetic diversity of PEDV. Our findings provide valuable information on PEDV evolution and underscore the need for ongoing surveillance of this economically important swine disease.