Cloning, expression and bioinformatics analysis of the duck TLR 4 gene

1. Toll-like receptors (TLRs) are type I transmembrane proteins that play an essential role in the innate immune system. Studies on the structure and function of TLRs can be applied to the development of new approaches to control diseases of humans and animals.

2. A 3432-bp cDNA encoding duck toll-like receptor 4 (duTLR4) was cloned from duck splenic lymphocytes using RT-PCR and rapid amplification of cDNA ends.

3. The encoded protein, which was predicted to contain 843 amino acids, had a molecular weight of 96·01?kDa and included an archetypal toll/interleukin-1 receptor domain, a transmembrane domain, and a distinctive arrangement of extracellular leucine-rich repeat regions similar to chicken TLR4, human TLR4, and mouse TLR4. The duTLR4 showed 82·1% amino acid sequence identity with previously described chicken TLR4, and 43·2–45·2% sequence identity with mammalian homologs.

4. RT-qPCR analysis indicated that the duTLR4 gene was strongly expressed in the liver, kidney, spleen, intestine, and brain.     

Functional characterisation of bovine TLR5 indicates species-specific recognition of flagellin

Mammalian toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and has been described to activate the innate immune system. To assess the role of bovine TLR5 (boTLR5) in the cattle system, we cloned and successfully expressed boTLR5 in human embryonic kidney (HEK) 293 cells, as indicated by quantitative PCR and confocal microscopy. However, in contrast to huTLR5-transfected cells, exposure of boTLR5-transfected cells to flagellin neither activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nor CXCL8 production. Subsequent comparison of the flagellin response induced in human and bovine primary macrophages revealed that flagellin did not lead to phosphorylation of major signalling molecules. Furthermore, the CXCL8 and TNFα response of primary bovine macrophages stimulated with flagellin was very low compared to that observed in human primary macrophages. Our results indicate that cattle express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. However, boTLR5 seemed to play a different role in the bovine system compared to the human system in recognizing flagellin, and other potentially intracellular expressed receptors may play a more important role in the bovine system to detect flagellin.     

猪生长激素促分泌素受体基因真核表达载体的构建及鉴定简

本试验旨在构建猪生长激素促分泌素受体（pGHS-R）真核表达系统,并瞬时转染人源胚胎肾细胞（HEK293T）观察其表达情况。以猪基因组为模板,通过剪接重叠延伸聚合酶链式反应（SOE-PCR）克隆出pGHS-R的编码区序列,插入真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1(+)/pGHS-R,酶切鉴定并测序,加myc标签,瞬时转染HEK293T细胞,用Western blotting鉴定该重组质粒是否能在真核细胞中表达相应的目的蛋白。结果显示,本试验成功扩增出pGHS-R编码序列,酶切和测序结果表明pcDNA3.1(+)-myc/pGHS-R构建正确,Western blotting方法证实转染的该质粒能在HEK293T细胞中正确表达目的蛋白。结果表明,本试验成功构建了pGHS-R真核表达载体,并正确表达蛋白,为进一步研究GHS-R的功能奠定了基础。     

Advanced molecular immunoassay system for immunobiotic lactic acid bacteria using a transfectant of Toll-like receptor 2

Toll‐like receptor 2 (TLR2) is a receptor for a variety of microbial components, and it also mediates activation signals in the cell relating to the innate immune system. In order to evaluate the precise molecular immunoregulation by various strains of lactic acid bacteria (LAB) via TLR2, the swine TLR2 (sTLR2)‐expressing transfectant was constructed using human embryonic kidney (HEK) 293 cells. It is demonstrated that intact immunobiotic LAB can induce immune responses through TLR2, and that different nuclear factor‐κB (NF‐κB) activities of various strains can be accurately detected by sTLR2‐expressing HEK293 cells. Furthermore, cellular activation of NF‐κB via TLR2 is reflected in enhanced binding and uptake of LAB. The sTLR2‐expressing HEK293 cells were also useful for characterizing the expression pattern of type I helper T (Th1) and type II helper T (Th2) cytokines by the stimulation of immunobiotic LAB. These results suggest that sTLR2‐expressing HEK293 cells may be useful in certain molecular immunoassay systems for producing new physiologically functional foods with intestinal immunomodulatory abilities, such as the maintenance of Th1/Th2 polarization.     

犬TfR胞外区密码子的优化、真核表达及与犬细小病毒VP2蛋白结合

为制备大量具有天然活性的犬胞外区可溶性转铁蛋白受体(sTfR),本试验通过密码子优化提高sTfR在真核细胞中的表达水平.利用RT-PCR方法从犬肝脏中扩增sTfR编码基因,依据该基因编码的氨基酸序列,参照人偏爱的密码子,对该基因进行密码子优化并由公司合成.利用peDNA3.1-CD5质粒分别构建野生型和密码子优化的sTfR基因真核表达载体,经磷酸钙介导使其在HEK293T细胞中进行表达,利用Western-blotting鉴定表达产物,通过ELISA检测重组犬sTfR蛋白与犬细小病毒VP2蛋白的结合活性.结果显示本试验扩增的犬sTfR基因与GenBank该基因序列的同源性为100%;通过在HEK 293T细胞中进行瞬时表达,结果显示密码子优化可以明显提高sTfR基因在HEK 293T细胞中的表达水平,提高了75%.同时表达的sTfR蛋白能够与犬细小病毒VP2蛋白进行特异结合,表明表达的重组sTfR蛋白具有天然活性.     

Construction of Eukaryotic Expression Vector pEGFP-E5-N1 of Bovine Papillomavirus Type 13 and its Expression in HEK293 Cells

The PCR technology was used to amplify the E5 gene according to the genome sequence of bovine papillomavirus type 13 (BPV-13) Hainan strain with the GenBank accession number KM258443.2.Then it was ligated into pEGFP-N1 vector.The recombinant plasmid pEGFP-E5-N1 was transfected into HEK293 cells with liposomes by transient transfection.The expression of fusion protein was identified by fluorescence microscope, flow cytometry and Western blotting.The results showed that the E5 gene was 135 bp with the 100% homology with the sequence deposited in GenBank.Bright green fluorescence could be seen in HEK293 cells transfected by pEGFP-E5-N1.The expressed fusion protein was about 32 ku.This experiment laid the foundation for further study of the functions of E5 gene in eukaryotic cells.     

牛乳状瘤病毒13型pEGFP-E5-N1真核表达载体的构建及其在HEK293细胞中的表达

基于海口市动物基因工程重点实验室获得的牛乳状瘤病毒13型(BPV-13)基因组序列信息(GenBank登录号:KM258443.2),以基因组为模板,扩增E5基因片段,将其连入pEGFP-N1质粒,构建重组质粒pEGFP-E5-N1,测序正确后瞬时转染HEK293细胞,应用荧光显微镜、流式细胞仪、Western blotting 鉴定融合蛋白的表达。结果显示,E5片段大小为135 bp;重组质粒pEGFP-E5-N1构建正确;荧光显微镜下观察转染重组质粒pEGFP-E5-N1的HEK293细胞,可见明亮的绿色荧光;流式细胞术分析结果显示,转染重组质粒pEGFP-E5-N1 48 h后,约55.59% 的细胞表达绿色荧光蛋白;Western blotting结果表明,表达的融合蛋白大小约为32 ku。本试验结果为下一步研究E5基因的作用机理奠定了基础。     

A potential test system for detecting contaminations by bacterial lipoproteins

Biological specimens are often contaminated with bacteria-derived products such as LPS or lipoproteins (LP), which trigger unwanted inflammatory responses in hosts. Whereas a contamination by LPS can be determined by various test systems, a contamination by LP can as yet not be determined. TLR4 and TLR2 are key components of the LPS and the LP receptor complex, respectively. It was tested in this study whether HEK293 cell stably transfected with bovine TLR2 have the ability to react to low concentrations of diacylated and triacylated synthetic LP. The stable cell lines we present here recognize low concentrations of synthetic LP resembling LP of different bacteria. Therefore, these cells are suitable to detect low contaminations present in probes. For example, HEK293 cells stably transfected with bovine TLR2 recognized an egg albumin preparation as contaminated, as evidenced by copious production of IL-8. In contrast, these cells did not respond to a highly purified human serum albumin (HSA) preparation used in the clinic but responded to HSA containing small amounts of diacylated synthetic LP. The TLR4 ligand LPS is often said to activate TLR2. Here we present evidence that LP contaminations are responsible for TLR2 activity. HEK293 cells stably transfected with bovine TLR2 and TLR1 (e.g. clone 1) did not respond to ultra-pure Escherichia coli LPS preparations but acquired responsiveness when stimulated with differently purified commercial LPS. Thus, the described system involving HEK293 cells stably transfected with bovine TLR2 and TLR1 is the first test system described attempting to measure a contamination by LP.     

Sequence and topological characterization of Toll-like receptor 8 gene of Indian riverine buffalo (Bubalus bubalis)

In this study, buffalo (Bubalus bubalis) Toll-like receptor 8 (TLR8) gene has been characterized by sequence analysis and detecting polymorphism. Complete ORF of buffalo TLR8 gene was amplified using the RNA isolated from spleen tissue, which was found to be 3,102 nucleotides long encoding a 1,033 amino acid protein. Buffalo TLR8 had 10 nucleotide changes as compared to other livestock species resulting in six unique amino acid changes, four of them lying within leucine-rich repeat (LRR) domains. As compared to cattle (Bos indicus and Bos taurus), out of fifteen cysteine residues, fourteen were conserved and Cys at position 521 was replaced by Arg. Nine of the LRR domains had no amino acid change as compared to cattle, whereas LRR-C-terminus had maximum, five amino acid changes. Sequence characterization of 12 riverine and swamp buffaloes revealed presence of four polymorphic nucleotides, two of them were non-synonymous, one synonymous and one site in 3′UTR. PCR-RFLP genotyping of non-synonymous SNP 2758A>G (ILeu920Val) in Toll–interleukin-1 receptor domain of 463 swamp and riverine buffaloes showed a higher frequency of allele A in swamp (95 %) as compared to riverine (9.84 %) buffaloes.     

猪β2肾上腺素能受体基因真核表达载体及突变体的构建及表达

试验旨在构建猪β₂肾上腺素能受体（β₂ adrenergic receptor,β₂AR）基因及其突变体真核表达载体,并鉴定其在HEK293T细胞中的表达。通过猪基因组DNA克隆猪β₂AR基因,利用同源重组技术将其连接至真核表达载体pcDNA3.1（+）,构建真核表达载体pcDNA3.1（+）-β₂AR,加入c-myc标签后命名为myc-pcDNA3.1（+）-β₂AR。以真核表达载体为模板构建突变体myc-pcDNA3.1（+）-β₂AR-D130N、myc-pcDNA3.1（+）-β₂AR-C285S和myc-pcDNA3.1（+）-β₂AR-D130N/C285S,并将构建的真核表达载体和突变体转染HEK293T细胞,利用Western blotting技术验证其表达。结果显示,猪β₂AR基因已正确重组入pcDNA3.1（+）载体中;经测序鉴定,猪β₂AR的第130位天冬氨酸成功突变为天冬酰胺,第285位半胱氨酸成功突变为丝氨酸。Western blotting检测结果证明所构建的表达载体均可在HEK293T细胞中表达。本研究成功构建了猪β₂AR野生型的真核表达载体及2个单点突变和1个双点突变的突变体,并验证其在HEK293T细胞中正常表达,为进一步研究猪β₂AR蛋白表达及其药理学活性奠定基础。     

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

牛lncRNA H19过表达载体构建

[目的]构建以及鉴定牛lncRNA H19过表达重组载体,为进一步探究lncRNA H19与miRNA 491和牛CART基因的互作关系奠定试验基础。[方法]NCBI获取牛lncRNA H19序列,经聚合酶链式反应(PCR)扩增,双酶切后载入pcDNA3.1-EGFP载体得到重组质粒。将pcDNA3.1-EGFP-H19、miRNA 491和CART 3种质粒共转染至HEK293T细胞,在细胞内反复扩增过表达之后,利用荧光定量技术检测pcDNA3.1-EGFP-H19的表达量。[结果]结果显示pcDNA3.1-EGFP-H19载体序列正确;293T细胞绿色荧光达50%,且强度适中,说明转染效果良好;lncRNA H19在293T细胞中显著表达。[结论]pcDNA3.1-EGFP-H19载体构建成功,试验将为后续探究lncRNA H19与miRNA 491和CART基因之间的互作关系创造试验条件。     

胆汁酸膜受体TGR5真核表达载体的构建及在细胞中的表达

试验旨在构建胆汁酸膜受体TGR5真核表达载体,转染293T细胞并在其中表达。用RT-PCR技术从胎盘组织中得到TGR5基因,克隆至真核表达载体pCMV-EGFP中。双酶切和测序鉴定正确后,将pCMV-EGFP-TGR5瞬时转染293T细胞,并采用实时荧光定量PCR和Western blotting技术检测TGR5的表达。结果显示,本试验构建了TGR5真核表达载体pCMV-EGFP-TGR5;转染293T细胞后,荧光显微镜观察到绿色荧光表达;实时荧光定量PCR检测TGR5表达量显著增加;Western blotting结果显示有目的蛋白表达。结果表明,真核表达载体pCMV-EGFP-TGR5构建成功,转染293T细胞后在基因、蛋白水平上均表达TGR5受体。     

Muscovy duck retinoic acid-induced gene I (MdRIG-I) functions in innate immunity against H9N2 avian influenza viruses (AIV) infections

Retinoic acid inducible gene I (RIG-I) is a cytosolic pattern recognition receptor that senses pathogen-associated molecular patterns (PAMPs). Muscovy duck (Cairina moschata) is a large duck different from other species of ducks, and is more susceptible to some microbial pathogens. In this study, the Muscovy duck RIG-I gene (MdRIG-I) was identified. Quantitative RT-PCR showed that MdRIG-I mRNA was widely expressed in different tissues, especially in those with mucosa. RIG-I null DF-1 cells transfected with DNA constructs encoding MdRIG-I or CARDs domain can activate IRF-3 and NF-κB to up-regulated activity of IFN-β promoter. The components of the signaling pathway downstream of RIG-I in mammalian cells including IRF-3, NF-κB, IFN-β and the IFN-stimulated genes Mx-1, PKR and MDA5 were significantly up-regulated in CARDs-overexpressing-DF-1 cells. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression in brain, spleen, lung and bursa were up-regulated in ducks challenged with H9N2 avian influenza virus (AIV), whose six internal genes were closely related to the H7N9 and H10N8 AIV. In vitro, DF-1 cells transfected with MdRIG-I plasmid can respond significantly to H9N2 AIV, evident through enhancement of IFN-β promoter activity and decreased virus titer. Altogether, these results indicated that MdRIG-I is a novel member of RLR gene family, engaging in the early stage of antiviral innate immunity.     

Muscovy duck reovirus infection rapidly activates host innate immune signaling and induces an effective antiviral immune response involving critical interferons

Muscovy duck reovirus (MDRV) is a highly pathogenic virus in waterfowl and causes significant economic loss in the poultry industry worldwide. Because the host innate immunity plays a key role in defending against virus invasion, more and more attentions have been paid to the immune response triggered by viral infection. Here we found that the genomic RNA of MDRV was able to rapidly induce the production of interferons (IFNs) in host. Mechanistically, MDRV infection induced robust expression of IFNs in host mainly through RIG-I, MDA5 and TLR3-dependent signaling pathways. In addition, we observed that silencing VISA expression in 293T cells could significantly inhibit the secretion of IFNs. Remarkably, the production of IFNs was reduced by inhibiting the activation of NF-κB or knocking down the expression of IRF-7. Furthermore, our study showed that treatment of 293T cells and Muscovy duck embryo fibroblasts with IFNs markedly impaired MDRV replication, suggesting that these IFNs play an important role in antiviral response during the MDRV infection. Importantly, we also detected the induced expression of RIG-I, MDA5, TLR3 and type I IFN in Muscovy ducks infected with MDRV at different time points post infection. The results from in vivo studies were consistent with those in 293T cells infected with MDRV. Taken together, our findings reveal that the host can resist MDRV invasion by activating innate immune response involving RIG-I, MDA5 and TLR3-dependent signaling pathways that govern IFN production.     

猪Mx1基因的克隆表达及抗病毒活性研究

本文应用(RT-PCR)技术,从猪瘟弱毒疫苗和Poly:IC共刺激7 h的PK15细胞的cDNA中扩增出编码Mx1蛋白的全长基因,并通过引物设计填补了PK(15)细胞系Mx1 cDNA序列中3’端11bp的缺失,成功获得Mx1完整基因的克隆。构建了重组表达质粒pRetroQ-sMx1,转染HEK293T细胞并表达Mx1蛋白,利用微量细胞病变抑制法测定其抗病毒活性。双酶切鉴定和核酸序列测定证实pRetroQ-sMx1真核表达质粒构建成功,转染HEK 293T细胞后,能够检测到绿色荧光,Western blot证实为目的蛋白。微量细胞病变抑制法测定重组蛋白具有一定的抗水疱性口炎病毒及猪繁殖与呼吸综合征病毒的活性。重组Mx1具有一定抗病毒生物功能,为进一步研究重组Mx1蛋白的活性以及Mx1抗病毒药物奠定了基础。     

山羊TLR2绿色荧光蛋白融合载体的构建

为了构建在哺乳动物细胞中表达的山羊TLR2绿色荧光蛋白融合载体,试验根据克隆到T载体上的山羊TLR2测序结果,设计1对不含终止子的引物,将山羊TLR2 PCR产物连接到pEGFP-N1载体上,用磷酸钙法转染293T细胞,并在荧光显微镜下观察。结果表明:重组质粒经酶切和测序鉴定正确,且在293T细胞中表达;融合蛋白发出绿色荧光,表明TLR2-EGFP主要分布在细胞膜上。说明试验成功地构建了pEGFP-TLR2-N1绿色荧光蛋白融合载体。