Molecular cloning and expression analysis of interferon-inducible transmembrane protein 1 in large yellow croaker Pseudosciaena crocea

In human cells, interferon-inducible transmembrane protein 1 (IFITM1) is a component of protein complexes involved in homotypic adhesion and the transduction of antiproliferative signals. Here, we reported the cloning of an IFITM1 homologue from the spleen of large yellow croaker Pseudosciaena crocea (LycIFITM1). The complete cDNA of LycIFITM1 is 734 nucleotides (nt) encoding a protein of 124 amino acids (aa), with a putative molecular weight of 13.6 kDa. The deduced LycIFITM1 protein is significantly homologous to interferon-inducible transmembrane proteins (IFITMs) in mammals and fish, and has the typical structural features of IFITMs, including two transmembrane domains (residues 43-63 and 90-112, respectively) and one intracellular domain between them (residues 64-89), as well as one conserved protein kinase C (PKC) phosphorylation site (residues 65-67, SIK). Phylogenetic analysis showed that LycIFITM1 formed a cluster with fish IFITM, reflecting a relative distant evolutionary relationship from mammals. LycIFITM1 gene was constitutively expressed in various tissues examined, such as gills, intestine, liver, kidney, heart, spleen, muscle and blood. Upon induction with poly(I:C), LycIFITM1 gene expression was obviously up-regulated in gills, kidney, heart and spleen at 24h after stimulation, suggesting that LycIFITM1 may be involved in the immune response induced by poly(I:C). Time course analysis using real-time PCR showed that the mRNA levels of LycIFITM1 in spleen and kidney were quickly up-regulated by poly(I:C) and reached the peak at 24h post-induction (48.7- and 280.4-fold mRNA increases in spleen and kidney, respectively). The results suggest that the IFITM1 homologue from large yellow croaker may represent a novel member of IFITMs family in fish.     

Characterization of tilapia (Oreochromis niloticus) viperin expression,and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.     

Equine herpesvirus type-1 modulates CCL2, CCL3, CCL5, CXCL9, and CXCL10 chemokine expression

Equine herpesvirus type 1 (EHV-1) is highly prevalent in horses and causes rhinopneumonitis, abortion, and encephalopathy. Studies on the related human herpes simplex virus and of murine models of EHV-1 suggest that chemokines play important roles in coordinating of innate and adaptive immune responses, and thus effective control of herpesvirus infection and prevention of severe clinical disease. Here, equine peripheral blood mononuclear cells (PBMC) were infected with one of three EHV-1 strains, which differ in pathogenicity (RacL11, NY03=abortogenic, Ab4=neurogenic). Changes in CCL2, CCL3, CCL5, CXCL9 and CXCL10 chemokine gene expression relative to non-infected PBMC were measured by real-time PCR. CXCL9 and CXCL10 gene expression was up-regulated 10h post infection and decreased to the level of non-infected cells after 24h. CCL2 and CCL3 were significantly down-regulated 24h post infection with NY03 and Ab4. CCL5 was up-regulated 24h after infection with RacL11. Ab4 infected PBMC had significantly lower expression of all chemokines except CCL2 24h post infection then RacL11 infected cells. While there was not a significant difference between NY03 and the other strains, there was a trend with each chemokine toward NY03 inducing less expression then RacL11 but more then Ab4. The data suggested that EHV-1 infection of PBMC induced up-regulation of inflammatory chemokines CCL5, CXCL9 and CXCL10, and down-regulation of chemotactic CCL2 and CCL3. The data also implies that different EHV-1 strains have varying effects on all five chemokines, with the nuropathogenic strain, Ab4, having the greatest suppressive potential.     

Laminar chemokine mRNA concentrations in horses with carbohydrate overload-induced laminitis

Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis.     

Activation of natural killer cells in newborn piglets by interferon induction

Natural killer (NK) cell activity in the peripheral blood lymphocytes (PBL) of newborn piglets, normally negligible, was stimulated by in vitro treatment with porcine type I interferon (IFN), and the NK activity of PBL from weaned piglets was augmented by the same treatment. Binding of the PBL to the PK-15 targets used in the single cell cytotoxicity assay for NK activity was not affected by age or by IFN treatment. When newborn piglets were treated with a single intravenous dose at 2 days of age of 0.5 mg/kg of polyinosinic:polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC), a synthetic IFN inducer, their IFN levels peaked at 6 h post-induction, and NK activity in their PBL peaked at 24 h post-induction at the level normally found in weaned piglets. The NK activity then declined until 7 days post-induction, when it increased again in a similar manner to that in untreated control piglets. Target-binding of the PBL was not affected by poly ICLC treatment of the piglets. Newborn piglets treated with poly ICLC and subsequently exposed to infection with transmissible gastroenteritis (TGE) virus showed a delay in onset of clinical signs of TGE compared with untreated control piglets. It was concluded that NK cells in newborn piglets can be activated by treatment of the piglets with poly ICLC, and that the presence of active NK cells is associated with some increase in resistance to challenge with TGE virus.     

Prevalence of Flavobacterium psychrophilum bacterial cells in farmed rainbow trout: Characterization of metallothionein A and interleukin1-β genes as markers overexpressed in spleen and kidney of diseased fish

The aim of the present study was to assess the prevalence of the flavobacteria within farmed trout and to quantify their bacterial burden. A total of 61 fish were sampled from seven farms, and were distributed in two groups: (1) visibly diseased fish suffering from the rainbow trout fry syndrome or the bacterial cold water disease caused by the bacteria Flavobacterium psychrophilum and (2) normally appearing fish. F. psychrophilum cells were titered by qPCR, targeting a specific area of the 16S rRNA gene in skin, muscle, gills, liver, spleen and kidney from all fish. The pathogen was detected in these organs whatever the health status, with titers ranging from 10⁴ to 6 × 10⁷ bacteria/g of tissue in normally appearing fish, thus showing they were bacterial carriers. Two organs allowed differentiation between diseased and normally appearing fish: spleen and kidney, with titers ranging from 10⁶ to 10⁷ bacteria/g of tissue in normally appearing fish vs 10¹¹ to 10¹² bacteria/g of tissue in diseased fish. No relationship was found between immunoglobulin M-like titer in plasma and health status. Gene expression analysis in fish organs revealed two genes that were markers of the bacterial infection: mt-a and il-1β genes encoding the metallothionein A and the interleukin1-β, respectively. These genes were both over-expressed in gills, liver, spleen and kidney of diseased fish. Four genes encoding immunity markers were down-regulated in spleen (a key organ implicated in immunity) of diseased fish: tgf-β, cd8-α, mhc2-β and igt, suggesting a weakened immune system in diseased fish.     

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.     

Assessment of bovine mammary chemokine gene expression in response to lipopolysaccharide, lipotechoic acid + peptidoglycan, and CpG oligodeoxynucleotide 2135

During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-α and IL-1β. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 μg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-α, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.     

犬圆环病毒Cap蛋白的生物信息学分析及原核表达

犬圆环病毒(CanineCV)是近年被发现的圆环病毒属新成员,获取CaineCV Cap蛋白,为研究Cap蛋白的功能以及抗原表位奠定了基础。本研究以CaineCV GX2017株基因组作为模板,使用PCR方法扩增出Cap蛋白的基因序列,对其进行生物信息学分析,并克隆至pET-28a原,进行原核表达。结果表明:GX2017的N端2~26位氨基酸存在一个核定位信号,同时含有3个B细胞表位(aa7~aa14;aa150~aa174;aa233~aa253);第134位氨基酸为N-糖基化位点,第169和第238位氨基酸为O-型糖基化位点;进化分析表明,本研究的CanineCV Cap蛋白基因序列与欧美株同源性较低,且处在不同的分支;SDS-PAGE结果显示,重组Cap蛋白在E.coli BL21(DE3)中不能正确表达,切除了NLS的d(1-26)Cap蛋白能在E.coli BL21(DE3)中大量表达;Western blot 分析表明,该重组蛋白能与Anti-His 标签抗体发生特异性反应。本研究成功构建了pET-d(1-26)Cap重组原核表达质粒,并在大肠杆菌中获得高水平表达,为进一步制备Cap蛋白的抗体奠定了基础。     

人工感染维氏气单胞菌对杂交鲟体内组织抗菌肽含量的影响

为探究抗菌肽在鲟鱼体内的表达规律,试验采用维氏气单胞菌人工感染杂交鲟后,采集不同时间段杂交鲟的肝脏、脾脏、心脏、肌肉、肠道、鳃6个组织样品,利用ELISA方法进行抗菌肽含量的检测。结果:(1)正常对照组杂交鲟6个组织中均有抗菌肽表达,在鳃和肠道组织中含量最高,在肝脏中含量最低。(2)与正常对照组比较,人工感染组杂交鲟6个组织中抗菌肽含量在不同时间段均明显升高,差异显著(P<0.05)。其中:在24 h和96 h鳃和肠道的抗菌肽含量差异极显著(P<0.01);在48 h肝脏、肌肉、心脏、脾脏的抗菌肽含量差异极显著(P<0.01)。结论:杂交鲟可能是通过抗菌肽来抵御细菌感染,这为杂交鲟细菌性疾病的防治提供了新思路。     

Induction and sequencing of Rousette bat interferon alpha and beta genes

Bats are considered to be natural reservoirs for several viruses of clinical importance, including rabies virus, Nipah virus, and Hendra virus. Type I interferons (IFNs) is an important part of the immune system in the defense against viral infection. To investigate the function of type I IFNs upon viral infection in bats, the nucleic acid, and amino acid sequences of Egyptian Rousette (Rousettus aegyptiacus) IFN-alpha and -beta were characterized. Sequence data indicated that bat IFN-alpha consists of 562-bp encoded 187-aa, and IFN-beta consisted of 558-bp encoded 186-aa. Phylogenetic analysis of the overall identity of IFN-beta shared the highest sequence homology with pig IFN-beta in both nucleotide and amino acid level. Stimulation of bat primary kidney cells (BPKCs) and bat lung cell lines, Tb-1 Lu, with polyinosinic-polycytidylic acid (poly(I:C)) or exogenous bat type I IFNs resulted in increased type I IFNs mRNA expression in BPKCs, but not in Tb-1 Lu. Characterization of the bat IFN-alpha and -beta genes allows understanding of the immune responses upon stimulation in different tissues, thus providing practical strategies for control and treatment of clinically important diseases. These results are important especially for the virus infection, and suggest that future molecular studies on virus infection experiment of bats in vitro will require careful consideration of the differences of type I IFN expression patterns in different cell types.     

鸡Toll样受体15单克隆抗体的制备及其初步应用

旨在制备鸡Toll样受体15 (ChTLR15)的特异性单克隆抗体(mAb),并初步应用.利用PCR技术扩增ChTLR15第162-386位氨基酸,克隆于载体pET-30a中进行诱导表达,获得高纯度的重组ChTLR15 (162-386 aa)蛋白.然后采用皮下多点注射方式将ChTLR15免疫6周龄的雌性BALB/c小...     

Granulocyte responses to experimental injection of live and formalin-killed bacteria in carp (Cyprinus carpio)

The objective of the study was to investigate the dynamics of changes in number of granulocytes in bacterial infections of carp (Cyprinus carpio). Carp were inoculated with non-pathogenic or pathogenic bacteria and changes in type I (neutrophils) and type II granulocyte (basophils/eosinophils) counts in kidney, circulating blood and peritoneal cavity were assessed. After the injection of non-pathogenic bacteria (Escherichia coli), the number of type I and II cells in blood increased after 6-12h, but returned to the control level after 24-48 h. In contrast, after the injection of pathogenic bacteria (Aeromonas hydrophila), the number of type I cells initially increased followed by an increase in the number of type II cells. The peak counts of type I and II cells were at 12 and 24h after the injection, respectively. When the fish were given serial-injections of formalin-killed bacteria at 12-h intervals, the type II cells also predominantly increased and remained at high levels, following the peak count of type I cells.     

Cloning of equine chemokines eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, mRNA expression in tissues and induction by IL-4 in dermal fibroblasts

We report the cloning of four equine CC chemokines, eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, which show high levels of identity with their respective homologous sequences in other species. Using a multiplex RT-PCR, we have studied the constitutive mRNA expression of these four CC chemokines in skin, lung, liver, spleen, jejunum, colon and kidney of normal adult horses and compared this data with the eosinophil counts in the same samples. We demonstrate that eotaxin mRNA is only expressed in jejunum and colon, where there are large numbers of eosinophils suggesting that eotaxin might be recruiting eosinophils in the normal digestive tract of the horse. MCP-1 and MCP-4 are expressed in all tissues whereas MCP-2 is only found in some samples of lung, spleen, liver and kidney. We also report the early induction (2h) of equine eotaxin and MCP-4, and the up-regulation of MCP-1 by interleukin-4 in dermal fibroblasts, suggesting these chemokines might be involved in equine skin allergic diseases.     

Gene expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli and Staphylococcus aureus in vitro

Postpartum Dysgalactia Syndrome (PDS) represents a considerable health problem of postpartum sows, primarily indicated by mastitis and lactation failure. The poorly understood etiology of this multifactorial disease necessitates the use of the porcine mammary epithelial cell (PMEC) model to identify how and to what extent molecular pathogen defense mechanisms prevent bacterial infections at the first cellular barrier of the gland. PMEC were isolated from three lactating sows and challenged with heat-inactivated potential mastitis-causing pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for 3 h and 24 h, in vitro. We focused on differential gene expression patterns of PMEC after pathogen challenge in comparison with the untreated control by performing microarray analysis. Our results show that a core innate immune response of PMEC is partly shared by E. coli and S. aureus. But E. coli infection induces much faster and stronger inflammatory response than S. aureus infection. An immediate and strong up-regulation of genes encoding cytokines (IL1A and IL8), chemokines (CCL2, CXCL1, CXCL2, CXCL3, and CXCL6) and cell adhesion molecules (VCAM1, ICAM1, and ITGB3) was explicitly obvious post-challenge with E. coli inducing a rapid recruitment and activation of cells of host defense mediated by IL1B and TNF signaling. In contrast, S. aureus infection rather induces the expression of genes encoding monooxygenases (CYP1A1, CYP3A4, and CYP1B1) initiating processes of detoxification and pathogen elimination. The results indicate that the course of PDS depends on the host recognition of different structural and pathogenic profiles first, which critically determines the extent and effectiveness of cellular immune defense after infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0178-z) contains supplementary material, which is available to authorized users.     

荧光定量PCR检测Meq基因在马立克氏病病毒感染过程中的动力学变化

为探讨马立克氏病病毒强、弱毒株致癌基因Meq在感染过程中的变化规律,本研究用超强毒株RB1B与疫苗株CV1988分别感染次代鸡胚成纤维细胞（chicken embryo fiber cell,CEF）,用荧光定量PCR方法检测Meq的变化规律。结果发现,疫苗株CV1988的Meq在感染细胞内持续上调,而超强毒株RB1B的Meq在感染细胞内72h前为逐渐上调,然后下调;动物感染试验结果发现,超强毒株RB1B的Meq在鸡胸腺和脾脏组织内均表现为感染d21出现一个表达高峰,而疫苗株CV1988的Meq在SPF鸡胸腺q-d14出现高峰,然后下降。有趣的是CV1988的Meq在脾脏组织中d14表达最低,然后持续升高。本研究为进一步研究Meq在马立克氏病病毒感染过程中的致瘤机理提供重要资料。     

Study on the Expression of FBP1 Gene in AA Broilers under Cold Stimulating Environment

In order to study of FBP1 gene expression under cold stimulating environment,75 AA broilers as research object were selected and randomly assigned to 5 groups,one of group was control group that raising in normal temperature,the remaining four groups were cold stimulation groups that raising temperature was 3 ℃ lower than the control group.The time of cold stimulation in four groups were 1,3,5 and 24 h,respectively.Cold stimulation started at 8 days of age,ended at 21 days of age.Broilers were slaughtered at 22 days of age and investigated FBP1 gene expression in each group.The results showed that the expression of FBP1 gene existed tissue specificity under cold stimulating environment.The relative expression level of FBP1 gene in liver was significantly higher than the other tissues in 1 h group (P< 0.05);The relative expression level of FBP1 gene in lung were significantly higher than the other tissues except of liver in 3 h group except for liver (P< 0.05);The relative expression level of FBP1 gene in liver were significantly higher than the other tissues in 5 h group (P< 0.05);The relative expression level of FBP1 gene in thymus were significantly higher than the other tissues in 24 h group (P< 0.05).FBP1 gene expression inside the heart was on the decline in the same organization different treatment groups as the rime of cold stimulating was increasing except for 3 h group.FBP1 gene expression of liver in 5 h group was significantly higher than other FBP1 gene expression in each treatment group.FBP1 gene expression of spleen was significant decreased in 1 h group,the other group extend a downward trend with the extension of cold stimulation time.FBP1 gene expression in lung was downward trend after rising first.FBP1 gene expression in kidney was significantly decreased in 24 h group,and the other groups had no obvious change.FBP1 gene expression of thymus in 24 h group was significantly higher than the other groups (P< 0.05),and there was no significant difference between control group and other four terament groups (P >0.05).