干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响

为探索干扰素诱导跨膜蛋白1（interferon-inducible transmembrane protein 1,IFITM1）对猪源冠状病毒复制的影响,本研究选用猪传染性胃肠炎病毒（Transmissible gastroenteritis virus,TGEV）高致病毒株及其宿主细胞PK15作为研究对象。正常PK15细胞接种TGEV后,实时荧光定量PCR检测感染后不同时间点PK15细胞中一些干扰素刺激基因（interferon-stimulated genes,ISGs）的mRNA表达水平;利用慢病毒表达系统构建稳定表达和稳定干扰IFITM1表达的PK15细胞系。将一段靶向干扰猪源IFITM1的shRNA序列及猪源IFITM1全长,分别插入pLKO.1-EGFP-Puro载体及pLVML-Myc-MCS-IRES-Puro载体中,分别构建出pLKO.1-IFITM1shRNA-EGFP-Puro及pLVML-Myc-IFITM1-IRES-Puro重组质粒。将重组质粒与慢病毒包装质粒共转染293FT细胞后获得带有目的基因的重组慢病毒,慢病毒侵染PK15细胞后用嘌呤霉素进行筛选,获得稳定表达及稳定干扰IFITM1表达的PK15细胞系,分别命名为PK15-IFITM1及PK15-IFITM1^-/-,并分别用实时荧光定量PCR、间接免疫荧光试验（IFA）及Western blotting检测IFITM1干扰效率及表达情况;TGEV接种PK15-IFITM1^-/-和PK15-IFITM1,实时荧光定量PCR测定细胞中TGEV的拷贝数。结果显示,PK15细胞接种TGEV后的48 h内,一些ISGs的mRNA水平均有所上升;PK15-IFITM1^-/-细胞系的干扰效率为70%,PK15-IFITM1细胞系表达成功;在PK15-IFITM1^-/-细胞系中,IFITM1的mRNA水平显著下调,促进了TGEV的复制。反之,在PK15-IFITM1细胞系中,TGEV的复制受到了抑制。但IFITM1的表达或缺失却不影响TGEV对PK15的吸附作用。总之,IFITM1对TGEV有显著的抗病毒作用,IFITM1不影响TGEV对PK15细胞的早期吸附,这为后续IFITM1抗冠状病毒机制的研究奠定了基础。     

猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示：该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到10^2.0 TCID₅₀/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。     

2020年新疆地区猪繁殖与呼吸综合征病毒抗体检测与分析

为了解2020年新疆地区猪繁殖与呼吸障碍综合征病毒（PRRSV）抗体水平及其消长规律,本试验采用间接酶联免疫吸附试验（ELISA）法对1 218份血清中PRRSV免疫抗体水平进行检测和分析。结果显示,PRRSV抗体平均阳性率为78.49%（956/1218）,高于国家规定标准（70%）。S/P的平均值为1.22±0.84,变异系数为69.45%。不同类别猪群抗体平均阳性率在59.19%~91.50%之间,有一定的差异。在调查的10个规模化养殖场中,9个场PRRSV免疫抗体阳性率达到国家标准。不同类别猪群PRRSV变异系数较高,需进一步对现有的免疫程序进行调整和完善,确保各猪群健康。     

2019—2020年新疆部分地区猪PCV2的抗体水平检测及分析

为了解2019—2020年新疆地区猪圆环病毒病2型（PCV2）抗体水平及其消长规律,本试验采用间接酶联免疫吸附试验（ELISA）方法对475份血清中PCV2免疫抗体水平进行检测和分析。结果显示,PCV2抗体平均阳性率为69.68%（331/475）,未达到国家规定标准（70%）。S/P的平均值为1.56。不同类别猪群抗体平均阳性率在42.50%~86.67%之间,有一定的差异。在调查的5个规模化养殖场中,仅有2个场PCV2免疫抗体阳性率达到国家标准。各场应根据抗体检测结果,进一步对现有的免疫程序进行调整和完整,确保各猪群健康。     

猪丹毒和猪肺疫混合感染综合诊治

疫病一直是生猪养殖中影响猪群健康的重要因素,受到疾病影响的猪群会给养殖户带来巨大的经济损失,严重阻碍养殖业的发展。该文将简要概述猪丹毒和猪肺疫的病理状况,分析猪丹毒和猪肺疫混合感染的发病特征和临床症状,并研究综合诊治猪丹毒和猪肺疫混合感染的方法,降低猪丹毒和猪肺疫混合感染对生猪养殖的影响。     

珠江三角洲地区生鲜农产品冷链网络化运输技术

文章主要是对我国的珠江三角洲地区的生鲜农产品冷链的物流方式进行探究,结合现在我国的生鲜农产品冷链运输技术的现状和发展前景进行大胆的预测,并提出我国的珠江三角洲地区的生鲜农产品冷链的运输技术的网络化的建议。     

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.     

Analysis on Differential Expression Genes in Porcine Aortic Vascular Endothelial Cells Induced by Apolipoprotein C Ⅲ

The aim of this study was to investigate the differential expression genes induced by ApoCⅢ,and study the function of ApoCⅢ.Porcine aortic vascular endothelial cells were successfully isolated using enzyme digestion,and then screened the differential expression genes induced by ApoCⅢ using the Solexa high-throughput sequencing technology.The results showed 647 differential expression genes,including 390 up-regulated genes and 257 down-regulated genes.The qRT-PCR results verified that the gene expression results from Solexa sequencing data were reliable.GO and Pathway analysis showed that the function of differential expression genes were related to immune response,cell apoptosis and death.These findings suggested that ApoCⅢ affected the physiological function of porcine aortic endothelial cells by the molecular pathways of inflammation,cell adhesion and apoptosis,which provided a theoretical basis for further understanding the molecular mechanisms of atherosclerosis caused by ApoCⅢ.     

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.     

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.