蓝舌病毒NS4基因真核表达对IFN-β信号通路基因mRNA表达的影响

通过构建蓝舌病毒(BTV)NS4基因真核表达载体pcDNA3.1-NS4-eGFP,转染HEK-293T细胞,利用Western blot及荧光显微镜分析NS4蛋白的表达与亚细胞定位特征;pcDNA3.1-NS4-eGFP转染的HEK-293T细胞添加20 HAU/mL仙台病毒(SeV)刺激后,qRT-PCR法分析NS4基因表达对SeV诱导的上游识别基因RIG-Ⅰ、MDA5、VISA、TBK1、IKKε、IRF3、TRAF3、TRAF6、IRF9、干扰素基因(IFN-α、IFN-β)以及干扰素刺激基因ISG15和USP18的mRNA表达水平的影响。在HEK-293T细胞内转染pcDNA3.1-NS4-eGFP质粒24 h后,分别添加20 HAU/mL SeV刺激24,48 h,qRT-PCR结果表明,细胞内表达NS4-EGFP后,RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15及IFN-β基因mRNA表达极显著下降,随着SeV诱导时间的延长,VISA、TBK1、IKKε、USP18基因mRNA表达差异呈不显著趋势。本研究成功构建BTV NS4基因真核表达载体pcDNA3.1-NS4-eGFP,NS4-EGFP融合蛋白在HEK-293T细胞中主要分布于细胞核周围及细胞核内。BTV NS4基因在HEK-293T细胞内的表达显著下调SeV诱导的IFN信号通路相关基因RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15和IFN-β的表达,为进一步探究NS4基因在BTV拮抗宿主细胞免疫应答中的机制奠定基础。     

Coexpression of avian influenza virus H5 and N1 by recombinant Newcastle disease virus and the impact on immune response in chickens

To analyze the contribution of neuraminidase (NA) toward protection against avian influenza virus (AIV) infection, three different recombinant Newcastle disease viruses (NDVs) expressing hemagglutinin (HA) or NA, or both, of highly pathogenic avian influenza virus (HPAIV) were generated. The lentogenic NDV Clone 30 was used as backbone for the insertion of HA of HPAIV strain A/chicken/Vietnam/P41/05 (H5N1) and NA of HPAIV strain A/duck/Vietnam/TG24-01/05 (H5N1). The HA was inserted between the genes encoding NDV phosphoprotein (P) and matrixprotein (M), and the NA was inserted between the fusion (F) and hemagglutinin-neuraminidase protein (HN) genes, resulting in NDVH5VmPMN1FHN. Two additional recombinants were constructed carrying the HA gene between the NDV P and M genes (NDVH5VmPM) or the NA between F and HN (NDVN1FHN). All recombinants replicated well and stably expressed the HA gene, the NA gene, or both. Chickens immunized with NDVH5VmPMN1FHN or NDVH5VmPM were protected against two different HPAIV H5N1 and also against HPAIV H5N2. In contrast, immunization of chickens with NDVN1FHN induced NDV- and AIV N1-specific antibodies but did not protect the animals against a lethal dose of HPAIV H5N1. Furthermore, expression of AIV N1, in addition to AIV H5 by NDV, did not increase protection against HPAIV H5N1.     

Host immune-related gene responses against highly pathogenic avian influenza virus infection in vitro differ among chicken cell lines established from different organs

Highly pathogenic avian influenza virus (HPAIV) induces acute disease in chickens causing high mortality and morbidity and is a major threat to poultry industries in Southeast Asian countries. The mechanisms of disease manifestation and host innate immune responses against HAPIV in chickens are not well understood. In this study, we examined virus replication and host gene expressions in four chicken cell lines in vitro to elucidate the impact of host innate immune responses against viral replication. It was demonstrated that viral replication efficiencies were different depending on the cell line. The viral replication appeared to be affected by the basal expression of IFN related genes. The expression of immune-related genes against the viral infection also varied in a cell line dependent manner. In non-immune derived cell lines, but not in immune derived cell lines, the expression of the CCL5 and CCL20 genes were induced by HPAIV infection. Reverse genetics HPAIV, with internal genes from avirulent avian influenza, reduced virus replication and affected immune-related gene expression in a cell line dependent manner. These results suggest the possibility that differential immune responses in different cell types in local tissues could modulate the consequences of HPAIV infection in chickens.     

lncRNA在乙脑病毒感染PK15细胞过程中的作用研究

本研究旨在初步探索乙脑病毒（JEV）感染PK15细胞后的增殖情况,筛选与病毒感染相关的lncRNA,并对其进行亚细胞定位及靶基因预测。通过免疫荧光试验来检测病毒结构蛋白E的表达情况,采用TCID₅₀法检测PK15细胞中病毒的增殖情况,利用实时荧光定量PCR检测病毒感染后lncRNA的表达水平,在NONCODE数据库对lncRNA进行亚细胞定位,通过starBase、NONCODE、KEGG等数据库对其进行靶基因预测和信号通路分析。结果显示,JEV感染PK15细胞后,24~36 h为病毒滴度指数增长期,感染后36 h病毒滴度已达10^-5.75 TCID₅₀/mL。PK15细胞在感染JEV 12 h后,lncRNA A、B、C表达水平均无显著变化（P>0.05）,lncRNA D表达水平极显著下降（P<0.01）;感染JEV 24、36和48 h后lncRNA A、B、C表达水平极显著上升（P<0.01）,lncRNA D表达水平极显著下降（P<0.01）。lncRNA A主要定位在胞质溶胶,lncRNA B在细胞核和细胞质中均有分布,lncRNA C主要定位在细胞质中,在细胞核中也有可能分布,lncRNA D可能在细胞内呈现广泛性分布。通过靶基因预测和信号通路分析,lncRNA A、B、C的靶基因主要为OAS1、OAS2、OASL、COX1等,lncRNA D的靶基因主要为DST、ND1、ND2、ND4等。信号通路分析发现lncRNA可能通过肿瘤坏死因子（TNF）、NF-κB和Toll样受体（TLR）等信号通路参与病毒感染后的增殖过程。本研究为进一步探索宿主细胞lncRNA对病毒增殖的影响奠定一定的基础。     

瑶山亚种树鼩ISG15蛋白表达及其多克隆抗体制备

【目的】克隆瑶山亚种树鼩干扰素刺激基因15(interferon stimulating gene 15,ISG15)基因,在大肠杆菌中高效表达并纯化、制备多克隆抗体,为其生物学应用及检测方法的建立奠定基础。【方法】提取瑶山亚种树鼩外周淋巴细胞总RNA,经RT-PCR扩增出ISG15基因,亚克隆到真核表达载体构建重组真核表达质粒pcDNA3.1-ISG15,并瞬时转染仓鼠肾细胞(baby hamster syrian kidney, BHK-21);同时亚克隆到原核表达载体pET-28a(+)构建重组表达质粒pET-28a-ISG15,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达树鼩ISG15蛋白;重组蛋白经镍离子亲和层析法纯化并免疫小鼠,获得鼠抗树鼩ISG15多克隆抗体,利用Western blotting和间接免疫荧光技术(IFA)检测其反应性。【结果】成功克隆瑶山亚种树鼩ISG15基因,并构建了其真核和原核表达载体,真核表达载体在BHK-21细胞中能高效表达;原核表达载体在大肠杆菌中30℃、0.5 mmol/L IPTG诱导6 h获得重组蛋白(分子质量22 ku),...     

Promoter identification and effect on activation of NF-κB of porcine ISG58

Interferon (IFN)-stimulated gene (ISG) 56 family (composed of ISG54, ISG56, ISG58, and ISG60) plays important roles in defense against viral infection in mammalian cells. Numerous studies have been conducted on ISG54, ISG56, and ISG60; however, little is known on ISG58. In the present study, the upstream sequence of porcine ISG58 gene was first characterized as functional promoter by luciferase reporter assay, and then two directly adjacent IFN-stimulated response elements (ISREs), one at ?206 to ?194 (ISRE-I) and a second one, directly upstream of this element at ?219 to ?207?bp (ISRE-II), were identified using the bioinformatics method. The subsequent site-directed deletion and transient transfection experiments showed the candidate ISREs are functional. ISRE-I works better than ISRE-II and synergistic cooperation exists between two ISREs. Additionally, the effect of porcine ISG58 on activation of NF-κB was analyzed using the dual-luciferase reporter assay. The results will contribute to revealing the role of ISG58 in immune response.     

Gene expression profile of HIRRV G and N protein gene vaccinated Japanese flounder, Paralichthys olivaceus during HIRRV infection

The glycoprotein (G protein) gene, but not the nucleocapsid protein (N protein) gene, of the hirame rhabdovirus (HIRRV) was previously shown to be highly effective in inducing a protective immune response in Japanese flounder (Paralichthys olivaceus) when used as a DNA vaccine. Our previous cDNA microarray analysis demonstrated that interferon-stimulated genes (ISGs) were strongly induced by the HIRRV G protein gene (pHRV-G) but not by the N protein gene (pHRV-N). However, the molecular basis for the difference in protective immunity between pHRV-G- and pHRV-N-vaccinated fish during HIRRV infection is still unclear. In this study, we use a DNA microarray to analyze differences of gene expression in pHRV-G- and pHRV-N-vaccinated fish during HIRRV infection. Microarray analyses showed substantial difference in gene expression patterns during HIRRV infection between fish vaccinated with pHRV-G and pHRV-N. In addition, genes having homology to mammalian T cell activation-related genes were up-regulated in the HIRRV G protein-vaccinated group.     

禽呼肠孤病毒感染DF1细胞后干扰素刺激基因在转录水平表达量的动态变化

为了解干扰素（interferon，IFN）和干扰素刺激基因在禽呼肠孤病毒（ARV）感染DF1细胞后的表达情况，试验将ARV病毒感染DF1细胞，观察细胞病变，收集感染后0、6、12、24、36、48、72、96 h的细胞样品，抽提反转录成cDNA，通过实时荧光定量PCR技术检测干扰素IFN-α和IFN-β及9种禽源常见干扰素刺激基因在感染后不同时间点在转录水平表达量的动态变化规律。结果显示，在ARV感染DF1细胞后，DF1细胞出现典型的细胞病变，感染后12 h病毒开始快速增殖，在36~96 h维持在较高的水平；IFN-α和IFN-β在转录水平的表达量在感染后均表现为显著下调（P<0.05；P<0.01）；IFI6、OAS、IFIT5、ISG12在转录水平表达量变化规律相似，均呈现显著上调表达（P<0.05；P<0.01），在感染后96 h达到峰值；其中IFIT5的上调幅度最大，感染后96 h的表达量是0 h的19.62倍（P<0.01）；而Mx、IFITM3、PKR、Viperin、ZAP的表达量变化规律相似，均表现为显著下调表达（P<0.05；P<0.01），其中Mx、IFITM3、Viperin的下调幅度较大，PKR和ZAP下调幅度很小。说明在ARV感染DF1细胞后，干扰素及多种干扰素刺激基因在转录水平呈现规律性变化，与病毒在DF1细胞中复制存在一定的联系。结果表明，ARV感染后可以诱导多种干扰素刺激基因的表达，这些干扰素刺激基因在抵御ARV病毒的入侵，抑制ARV的复制、释放及病毒的清除中发挥着重要作用。本研究为今后深入研究ARV的致病机理和宿主的抗病毒免疫应答提供了参考。     

Innate sensing of viruses by pattern recognition receptors in birds

Similar to mammals, several viral-sensing pattern recognition receptors (PRR) have been identified in birds including Toll-like receptors (TLR) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR). Avian TLR are slightly different from their mammalian counterparts, including the pseudogene TLR8, the absence of TLR9, and the presence of TLR1La, TLR1Lb, TLR15, and TLR21. Avian TLR3 and TLR7 are involved in RNA virus recognition, especially highly pathogenic avian influenza virus (HPAIV), while TLR15 and TLR21 are potential sensors that recognize both RNA viruses and bacteria. However, the agonist of TLR15 is still unknown. Interestingly, chickens, unlike ducks, geese and finches, lack RIG-I, however they do express melanoma differentiation-associated gene 5 (MDA5) which functionally compensates for the absence of RIG-I. Duck RIG-I is the cytosolic recognition element for HPAIV recognition, while chicken cells sense HPAIV through MDA5. However, the contributions of MDA5 and RIG-I to IFN-β induction upon HPAIV infection is different, and this may contribute to the chicken’s susceptibility to highly pathogenic influenza. It is noteworthy that the interactions between avian DNA viruses and PRR have not yet been reported. Furthermore, the role for avian Nod-like receptors (NLR) in viral immunity is largely unknown. In this review, recent advances in the field of viral recognition by different types of PRR in birds are summarized. In particular, the tissue and cellular distribution of avian PRR, the recognition and activation of PRR by viruses, and the subsequent expression of innate antiviral genes such as type I IFN and proinflammatory cytokines are discussed.     

m6A甲基化在鸡肌肉生长发育中的表达研究

试验旨在研究RNA m6A修饰相关基因去甲基化酶Alk B同源蛋白5（Alk B homologue 5，ALKBH5）、去甲基化酶肥胖相关蛋白（fat mass and obesity-associated protein，FTO）、甲基转移酶样蛋白3（methyltransferase like 3，METTL3）、甲基转移酶样蛋白14（methyltransferase like 14，METTL14）和成肾细胞瘤1-结合蛋白（Wilms’tumor 1-associating protein，WTAP）在鸡骨骼肌发育过程中的表达，分析其与骨骼肌m6A甲基化水平的相关性。首先，利用实时荧光定量PCR技术检测m6A甲基化相关基因在金茅花鸡12（E12）、14（E14）、16（E16）、18（E18）胚龄和1日龄腿肌和胸肌组织中mRNA表达水平，以及其在鸡成肌细胞50%、100%增殖期和1、2、3、4、5 d分化期的mRNA表达水平；随后，利用m6A甲基化试剂盒检测金茅花鸡E12和1日龄腿肌和胸肌组织中m6A甲基化修饰水平，与m6A甲基化相关基因表达水平进行相关性分析。结果显示，m6A去甲基化基因ALKBH5和FTO mRNA表达水平在骨骼肌发育过程中显著上调（P<0.05），即在E12、E14低表达，E16、E18逐渐上调，1日龄达到最高。m6A甲基化写入基因METTL14、METTL3和WTAP mRNA表达水平在E12、E14、E16逐渐上升，E18下降，随后至1日龄表达量回升。在细胞增殖过程中，ALKBH5、FTO、METTL14、METTL3和WTAP基因表达均上调；在细胞分化过程中ALKBH5和FTO基因表达水平显著上调（P<0.05），在分化第5天达到最高。METTL14、METTL3和WTAP基因mRNA表达水平在细胞诱导分化的1、2、3、4 d表达量呈下降趋势，而在诱导分化的第5天有所回升。甲基化水平检测结果显示，腿肌和胸肌m6A甲基化水平变化趋势一致，均在胚胎发育过程中显著下降（P<0.05），至1日龄达到最低。相关性分析结果显示，鸡骨骼肌RNA m6A甲基化水平与m6A去甲基化修饰基因ALKBH5、FTO mRNA表达水平呈显著负相关（P<0.05）。综合以上试验结果，推测m6A甲基化修饰与鸡骨骼肌发育相关，而去甲基化基因ALKBH5、FTO可能通过调控RNA m6A甲基化水平，影响鸡骨骼肌发育。本研究结果为进一步研究m6A甲基化修饰调控鸡骨骼肌生长发育的功能和分子机制提供理论依据。     

Identification and characterization of Japanese flounder, Paralichthys olivaceus interferon-stimulated gene 15 (Jf-ISG15)

Previously, using cDNA microarray analysis, we demonstrated that an EST clone of Japanese flounder (Paralichthys olivaceus) with homology to mammalian interferon-stimulated gene 15 (ISG15) was strongly induced by treatment with DNA vaccine encoding the glycoprotein gene of Hirame rhabdovirus (HIRRV). In this study, we conducted molecular cloning and expression analysis of the Japanese flounder ISG15 (Jf-ISG15). Jf-ISG15 encoded two exons. The first exon was non-coding, while the second exon encoded a protein of 158 amino acids. The coded protein has two tandem ubiquitin-like domains with a carboxyl-terminus conjugation motif “LRLRGG”. Phylogenetic analysis revealed an evolutionary relationship among Jf-ISG15, mammalian and fish ISG15 orthologues. The interferon-stimulated response element (ISRE) sites were conserved among DNA sequences of Jf-ISG15 and mammalian ISG15 promoter regions. An RT-PCR analysis of healthy tissues showed that Jf-ISG15 mRNA was notably strongly expressed in gills, PBLs and spleen. Expression of Jf-ISG15 was strongly induced by poly-I:C treatment in head-kidney cells, peripheral blood leukocytes (PBLs) and spleen cells, and by HIRRV infection in kidney of juvenile fish suggesting that Jf-ISG15 plays a role in fish antiviral response.     

Amplification,Sequence Analysis and Tissue Expression Detection of FUT2 Gene Coding Regions of Dongchuan Pigs

Porcine alpha (1,2) fucosyltransferase (FUT2) gene was importance in glycosphingolipid biosynthesis-globo series, potentially played a regulatory role during Escherichia coli (E. coli) F18 infection process in weaned piglets. In order to explore sequence structure of porcine FUT2 gene and its biological function, this test amplified FUT2 gene CDS sequence of Dongchuan pigs by PCR, forecasted and analyzed the protein sequences and functional regions of FUT2 gene, its expression level was detected in 11 tissues of 8 Dongchuan weaned piglets in 35 days old at the meantime. The results showed that the CDS sequence of FUT2 gene was 1 023 bp, which encoded 340 amino acids. FUT2 protein was fat-soluble hydrophilic protein, which the structure was not stable, including a transmembrane helix structure, but without signal peptide that suggested the FUT2 protein was a membrane protein;FUT2 protein included 2 N-glycosylation sites (No. 185 and No. 305 amino acids), without O-glycosylation sites, there were 14 potential phosphorylation sites, included 6 Ser, 2 Thr and 6 Tyr, analyzing the functional regions found that the FUT2 protein had a superfamily of conserved domains:FUT1-FUT2-like (58-319 amino acids). The phylogenetic tree result showed that the relative relationship between swine and cattle was relatively close, but was distant from chimpanzee, human, mouse and rat. FUT2 gene was expressed in all 11 tissues of Dongchuan weaned piglets, there were higher expression in digestive tract and immune tissues. The present results suggested that FUT2 gene might play a role to resistance to E. coli F18 in weaned piglets, and might indirectly against E. coli F18 through the synthesis of fucosyltransferase.     

东串猪FUT2基因CDS扩增、序列分析及组织表达谱检测

猪α-（1,2）岩藻糖转移酶2（FUT2）基因为鞘糖脂生物合成-球系列通路中的重要基因,可能在断奶仔猪抵抗大肠杆菌F18侵染过程中发挥着调控作用。为探究猪FUT2基因的序列结构及其生物学功能,试验采用PCR扩增得到地方猪品种东串猪FUT2基因的CDS全序列,进而预测和分析FUT2基因的蛋白质序列及其功能区域,同时对其在8头35日龄东串断奶仔猪11个组织中的表达水平进行检测与分析。结果显示,FUT2基因的CDS序列全长为1 023 bp,共编码340个氨基酸,FUT2蛋白为脂溶性的亲水蛋白,蛋白结构不稳定,该蛋白存在1个跨膜螺旋结构,但不存在信号肽,表明FUT2蛋白为膜蛋白;FUT2蛋白存在2个N-糖基化位点（185和305位氨基酸）,无O-糖基化位点,此外该蛋白还存在14个潜在的磷酸化位点,包括6个Ser、2个Thr和6个Tyr,对其功能区域进行分析发现,FUT2蛋白存在1个超级家族保守结构域：FUT1-FUT2-like（58-319位氨基酸）;系统进化树结果显示,猪与牛的亲缘关系相对较近,与人、黑猩猩、大鼠和小鼠等亲缘关系相对较远;FUT2基因在东串断奶仔猪11个组织中均有表达,在消化道和免疫组织中表达水平较高。试验结果推测FUT2基因在断奶仔猪抵抗大肠杆菌F18中可能具有一定的作用,且可能是通过合成岩藻糖转移酶间接发挥其抵抗大肠杆菌F18的作用。     

禽致病性大肠杆菌Hcp2b对雏鸡气管黏膜细胞因子-细胞因子受体相互作用通路的影响

T6SS(type Ⅵ secretion system)是革兰阴性菌中常见的一种分泌系统,其效应蛋白Hcp2b作用机制迄今仍未明晰.本研究以禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)Hcp2b蛋白为研究主体,旨在探究Hcp2b蛋白在APEC感染鸡气管黏膜过程中发挥...     

Characterisation of Mycoplasma gallisepticum strains involved in respiratory disease in pheasants and peafowl

Two cases of Mycoplasma gallisepticum infection in different avian species in backyard gamebird operations in Slovenia were investigated. In the first case, M gallisepticum was associated with severe respiratory disease with almost 20 per cent mortality in pheasants, whereas the infection was less pathogenic for chickens and turkeys reared at the same site. The M gallisepticum isolates from pheasants had a unique pMGA gene sequence containing a repeat of 12 nucleotides, and they contained only small amounts of the cytadhesins MGC1 and MGC3 and no PvpA protein. However, they expressed some typical M gallisepticum proteins and several proteins which were immunogenic for pheasants, chickens and turkeys. A strain of M gallisepticum isolated from the sinus of a pheasant was highly pathogenic for chicken embryos. In the second case, the M gallisepticum strain that was associated with respiratory disease and mortality in peafowl also affected chickens. M gallisepticum strain ULB 992 was isolated from the infraorbital sinus of a dead peafowl. The ULB 992 strain synthesised a small amount of MGC3, a truncated form of MGC1 and lacked PvpA. However, it expressed several proteins which were immunogenic for the birds infected with M gallisepticum at both gamebird operations.     

利用酵母双杂交技术筛选和鉴定非洲猪瘟病毒E120R蛋白的互作宿主蛋白

【目的】 探讨非洲猪瘟病毒（African swine fever virus，ASFV）衣壳蛋白E120R在病毒感染中的作用，确定与E120R蛋白互作的宿主蛋白。【方法】 将ASFV分离株CADC_HN09株E120R基因合成并克隆于pGBKT7表达载体，获得pGBKT7-E120R诱饵质粒，同时构建E120R基因截短表达质粒pGBKT7-E120R-1（1－61位氨基酸）和pGBKT7-E120R-2（62－122位氨基酸）。经毒性和自激活性检测后，通过酵母双杂交技术对骨髓巨噬细胞（BMDMs） cDNA均一化酵母文库进行初步筛选，以获得与ASFV E120R蛋白互作的蛋白。通过NCBI数据库进行序列比对并经免疫共沉淀试验验证，确定互作的宿主蛋白。筛选的宿主蛋白经webgenstal在线分析网站初步进行GO功能和KEGG通路富集分析，以确定所筛选的宿主蛋白参与的生物过程与信号通路。【结果】 诱饵质粒pGBKT7-E120R-2无毒性和自激活性，可用于文库筛选。通过酵母双杂交系统从BMDMs cDNA均一化酵母文库中初步筛选到46个阳性克隆并进行回转验证，经NCBI数据库序列比对分析共获得29个宿主蛋白。免疫共沉淀试验结果显示，多聚胞嘧啶结合蛋白2（PCBP2）和干扰素刺激基因15（ISG15）均与E120R蛋白存在互作。GO功能富集分析表明，所筛选的宿主蛋白可参与代谢过程、生物调节、应激反应等生物过程；KEGG通路富集分析表明，这些宿主蛋白可参与抗原呈递、铁死亡和坏死性凋亡等多条信号通路。【结论】 ASFV E120R蛋白可与宿主免疫应答、细胞死亡等信号通路相关的多种宿主蛋白互作，为进一步研究ASFV E120R蛋白在病毒感染过程中的作用提供了重要理论依据。     

PRV感染三叉神经节细胞对PI3K/Akt信号通路的影响及其调控凋亡

以小鼠三叉神经节(TG)原代细胞为基础,应用实时荧光定量PCR(q-PCR)、Western blot等方法检测伪狂犬病病毒(pseudorabies virus,PRV)(MOI=1)感染TG细胞后不同时间点PI3K、Akt基因的转录水平和蛋白表达情况.PRV感染TG细胞后,利用PI3K特异性抑制剂LY294002处...     

凋亡相关斑点样蛋白敲除PK-15细胞系的建立及对PRV感染的影响

本试验旨在研究凋亡相关斑点样蛋白(ASC)的基因敲除对猪伪狂犬病病毒(PRV)感染PK-15细胞的影响.以慢病毒介导的CRISPR/Cas9基因编辑技术,构建猪肾上皮细胞(PK-15)A SC基因稳定敲除细胞系,通过T7核酸酶检测靶基因的敲除效率;CCK-8试剂盒检测PK-15敲除ASC基因对细胞增殖的影响;采用流式细...     

S100A12在胸膜肺炎放线杆菌诱导猪肺泡巨噬细胞炎性细胞因子表达及吞噬中的作用

试验旨在探究S100A12在胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）诱导猪肺泡巨噬细胞（porcine alveolar macrophages，PAM）炎性细胞因子表达及吞噬中的作用。PAM细胞接种APP菌株后，用实时荧光定量PCR检测感染后不同时间点炎性细胞因子和S100A12的mRNA表达水平；构建S100A12重组蛋白表达载体并进行重组蛋白的表达纯化，用MTT法检测其对PAM的细胞毒性，实时荧光定量PCR检测不同浓度（0、5、50和500 ng/mL） S100A12重组蛋白对PAM炎性细胞因子mRNA表达水平的影响；构建S100A12干扰质粒，用Western blotting、实时荧光定量PCR及流式细胞术检测其干扰效率及其对APP感染PAM后炎性细胞因子mRNA表达水平和细胞凋亡的影响；用平板计数法检测S100A12表达对APP的黏附侵袭及其在PAM细胞内存活的影响。结果表明，APP感染促进PAM中IL-6、IL-8、IL-18、IL-1β和TNF-α等炎性细胞因子表达的同时也促进S100A12的表达，且该表达随APP感染剂量增大及时间的延长均显著或极显著增加（P<0.05；P<0.01）。5、50和500 ng/mL重组蛋白S100A12对PAM均没有毒性。5和50 ng/mL的重组蛋白S100A12均可显著促进PAM中IL-6、IL-8、TNF-α、IL-1β、IL-21和IL-5等炎性细胞因子的表达（P<0.05），而高浓度的S100A12重组蛋白则对上述炎性细胞因子表达无影响（P>0.05）。干扰质粒可成功阻断S100A12蛋白的表达，与转染shNC的对照组相比，在APP诱导下转染shS100A12干扰质粒的PAM中细胞因子IL-6、IL-8、IL-1β和IL-5的转录水平显著或极显著降低，且细胞凋亡率也显著或极显著增加（P<0.05；P<0.01）。进一步研究发现，干扰PAM中S100A12蛋白的表达可显著增强APP对PAM的黏附侵袭能力及其在PAM内的存活能力（P<0.05），相反，体外添加S100A12重组蛋白则显著抑制APP对PAM细胞的黏附侵袭及其在PAM内的存活（P<0.05）。以上研究表明，S100A12具有增强APP感染后PAM炎性细胞因子的表达、抑制APP诱导的PAM凋亡和降低APP对PAM的黏附侵袭及其在PAM内存活的作用，为进一步揭示APP感染过程中S100A12对PAM调控作用及机制奠定了基础。     

神经肽S及其受体对伪狂犬病病毒感染的体外作用研究

为了解神经肽S（NPS）及其受体（NPSR）在伪狂犬病病毒（PRV）感染过程中的作用,本试验采用细胞培养、RT-PCR、实时荧光定量PCR及shRNA技术研究NPS和NPSR在PRV体外感染过程中的表达变化,以及NPS和NPSR对病毒基因和相关细胞因子的变化。RT-PCR和实时荧光定量PCR结果显示,PRV感染培养的小鼠胚胎成纤维细胞系（3T3细胞）后12和18 h,NPS和NPSR mRNA的表达水平极显著上升（P<0.01）;通过shRNA技术稳定干扰3T3细胞中NPSR表达后,PRV gE基因的表达极显著下调（P<0.01）;添加外源性NPS可显著增强PRV感染的3T3细胞PRV gE基因及细胞因子白介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的表达水平（P<0.05）;添加NPSR抑制剂后,则极显著抑制细胞中PRV gE基因和细胞因子IL-6和TNF-α的表达水平（P<0.01）,该作用效果与NPS的作用效果相反。上述结果表明,降低NPSR的表达能有效抑制PRV在3T3细胞上的复制,说明NPSR是PRV有效感染3T3细胞所必需的重要因子;外源性NPS可通过调节炎症细胞因子的表达而加剧PRV感染诱导的炎症反应。本研究结果为深入探索NPS和NPSR在PRV感染动物体内的调节作用奠定了基础。