长毛兔和獭兔白细胞介素-4基因的序列分析与真核表达

应用反转录—聚合酶链式反应（RT-PCR）技术,在国内首次从ConA诱导培养的长毛兔和獭兔外周血淋巴细胞总RNA中扩增出IL-4基因。将扩增基因克隆入pMD18-T载体,经序列测定分析表明,长毛兔和獭兔IL-4基因全长均为441 bp,两序列间碱基组成无差异（登录号：EF606761和EU639687）。长毛兔和獭兔IL-4基因编码147个氨基酸,其中前24个氨基酸组成信号肽,余下的123个氨基酸组成成熟肽。与已报道的犬、猫、人、猪、牛、羊、马、大熊猫和鼠的IL-4核苷酸同源性分别为61.8%、63.4%、69.8%、65.1%、64.1%、64.1%、63.1%、57.0%、60.5%,推导的氨基酸同源性分别为47.2%、50.0%、53.6%、52.1%、48.3%、46.9%、52.1%、43.7%、43.0%。将测序正确的阳性质粒采用脂质体法转染COS-7,用RT-PCR鉴定转染细胞中IL-4基因的表达,并通过MTT法检测到表达的IL-4蛋白具有一定的生物学活性。     

Cloning and expression of goat interleukin-18 gene

We isolated and sequenced a 480 bp cDNA encoding mature goat interleukin-18 (gIL-18) from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The gIL-18 gene was cloned into pET32a (+) vectors and sequenced. Nucleotide sequence of gIL-18 shares high homology with cattle. Fusional expression with pET32a (+) of gIL-18 of about 38kD was obtained by SDS-PAGE analysis after induction by IPTG in the E. Coli BL21 expression system. The recombinant protein can induce IFN-gamma production in PBMC. The IL-18 mRNA was constitutively detected in goat alveolar macrophages with or without LPS, While, enhanced expression was detected in splenocytes and liver cells if treated by LPS, and can be weakly detected in Peripheral blood mononuclear cells (PBMCs) treated by activators. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues.     

Immunomodulation of caprine lentiviral infection by interleukin-16

Interleukin-16 (IL-16) is a proinflammatory cytokine produced by a variety of cells including lymphocytes, macrophages, mast cells, and eosinophils. We have shown in our previous studies increased expression of IL-16 mRNA and protein in caprine arthritis-encephalitis virus (CAEV)-infected goats blood. In this study, we determined the immunomodulatory effects of IL-16 in vitro using cells derived from CAEV infected and uninfected goats. Human recombinant IL-16 (rhIL-16) significantly increased chemotaxis of peripheral blood mononuclear cells (PBMCs) of both control and CAEV-infected goats. Pretreatment of PBMC with anti-goat CD4 monoclonal antibody inhibited IL-16-induced chemotaxis of PBMC of control and infected goats suggesting that IL-16 exerts its action in goats primarily by binding to CD4. The CAEV proviral DNA was less in caprine monocytes treated with rhIL-16 infected in vitro with CAEV. These data suggest inhibitory effect of IL-16 on viral integration. Flow cytometric studies indicated a trend toward IL-16-induced increased expression of lymphocyte activation markers. Combined with our previously reported data, these experiments suggest that increased IL-16 expression during CAEV infection may inhibit viral integration.     

Quantitative Analysis of Interleukin-6 Expression in Porcine Spleen Cells and Alveolar Macrophages using Real-Time PCR

Interleukin-6 (IL-6), a multifocal cytokine produced by lymphoid and non-lymphoid cells, regulates immune responses, acute-phase reactions against bacterial infections, and haematopoiesis. After cloning and sequencing of porcine IL-6, the expression pattern of porcine IL-6 mRNA was evaluated through real-time RT-PCR using porcine immune cells (spleen cells and alveolar macrophages) following stimulation with LPS. The sequence has been reported to GenBank with Accession no. AF 518322. The nucleotide sequence was different at the 89th and 205th positions in comparison with M80258, but only at the 205th with M86722. Comparison of porcine IL-6, Accession no. AF 518322, with IL-6 of human, canine, ovine, and mouse showed homologies of 78%, 81%, 82% and 73% in nucleotide sequence and 42%, 69%, 61% and 42% in amino acids. Expression of IL-6 mRNA was induced by stimulation with LPS. IL-6 mRNA expression in alveolar macrophages peaked at 2 h and decreased sharply to control levels at 4 h, whereas it peaked at 14 h and decreased at 24 h in spleen cells after stimulation with LPS (1 microg/ml). These results suggest that IL-6 mRNA expression in porcine immune cells is cell-type specific and the results of this study could be used as the basis for research on the porcine immune system.     

Cloning,expression, and tissue distribution of bovine interleukin-21

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.     

Expression of rabbit interleukin-4 and characterization of its biologic activity on T and B-cells

The purpose of the current study was to express recombinant rabbit IL-4 (rRbIL-4) and to characterize its biological activity. The cDNA of RbIL-4 was cloned into an insect cell expression vector that allowed for constitutive expression in Sf9 cells and incorporated a 6-histidine tag on the recombinant protein for purification. The purified protein corresponded to the predicted size of rRbIL-4 and was recognized by an anti-human IL-4 antibody in immunoblotting. As shown for IL-4 from other species, a dose-dependent proliferative response was observed in T-lymphoblasts cultured with rRbIL-4. rRbIL-4 also induced increased expression of MHC class II molecules on the surface of rabbit B-cells in a dose-dependent manner. These results indicate that we have produced recombinant rabbit IL-4 that exhibits expected biological activity on rabbit B and T-cells.     

山羊溶酶体α-AMA基因的克隆、生物信息学及组织表达谱分析

本研究旨在对山羊溶酶体α-甘露糖苷酶(α-AMA)基因进行组织表达谱和生物信息学分析。参考牛α-AMA基因序列设计引物,采用PCR技术克隆山羊α-AMA基因序列,并利用荧光定量RT-PCR进行组织表达谱分析以及进行生物信息学预测。首次获得了山羊α-AMA基因,含有完整CDS编码区3 000bp,编码999个氨基酸,其中前50个氨基酸为信号肽序列。其编码区的核苷酸序列和预测氨基酸序列与牛的α-AMA相似性最高,分别为95.93%和94.79%。组织表达谱分析表明α-AMA在山羊各组织均不同程度的表达,其中在肺脏、肝脏、小脑表达量较高。生物信息学预测发现,α-AMA蛋白属于糖苷水解酶38家族成员,有2个保守的结构域,存在9个N-糖基化位点。SWISS-MODEL同源建模山羊α-AMA具有良好的可信度。本研究为探讨酶的作用机理及疯草解毒剂的研制提供了理论依据。     

Expressed sequence identification and characterization of the cDNA for interleukin-4 from the mitogen-stimulated lymphoid tissue of a marsupial, Macropus eugenii

Very few cytokines that are important to the understanding of T helper cell function are characterized in marsupials. Expression of a 645 bp cDNA product that codes for a predicted Interleukin-4 peptide of 157 amino acids was detected in the lymph node tissues of Macropus eugenii, the tammar wallaby. Using Rapid Amplification of cDNA Ends, both 5'- and 3'-untranslated regions were identified and a polyadenylation signal and three mRNA instability motifs associated with secreted cytokine molecules were also present. The translated cDNA sequence has a putative signal peptide of 24 amino acids, a predicted secondary structure that is consistent with the short-chain alpha-helical cytokine family and 82% conservation of residues associated with the Interleukin-4 family sequence motif. Comparisons of wallaby nucleotide and predicted peptide sequences with the coding domains of other vertebrate species demonstrate the diversity within this gene family; with nucleotide and amino acid identities of 74% and 59% with opossum, 52% and 32% with human and 38% and 19% with chicken homologues respectively. Despite these differences in sequence conservation, the putative Macropus eugenii Interleukin-4 mature peptide contains conserved structural motifs and predicted receptor-binding residues that suggest that it may retain functional properties associated with this important Th2 cytokine in other mammals.     

Molecular cloning, expression and characterization of an interleukin 27 p28 homolog in pig (Sus scrofa)

IL-27 is the newest member of the IL-12 cytokine family and plays an important role in the immune regulation. It is composed of two subunits, p28 and EBV-induced gene 3(EBI3). Although human and mouse IL-27 p28 genes have been cloned, pig IL-27 p28 gene has not ever been reported. In the present study, we have cloned and characterized the full-length cDNA of IL-27 p28 from pig. The open reading frame of pig IL-27 p28 gene is 720 bp, which encodes a protein of 239 amino acids with a predicted molecular mass of 26.6 kDa. The deduced amino acid sequence of pig IL-27 p28 showed a high degree of homology to human (63%) and mouse (58%). It was a 4-helix cytokine and belonged to 4-helix cytokine superfamily. Pig IL-27 p28 has one transmembrane region, one signal peptide, and one N-glycosylation site, two Protein kinase C phosphorylation sites, three Casein kinase II phosphorylation sites and one N-myristoylation site. For the expression of pig IL-27 p28 protein in a eukaryotic expression system the recombinant plasmid was constructed. The expression of pig IL-27 p28 in mammalian cells were confirmed by flow cytometry analysis, immunofluorescence and Western blot. The analysis also confirmed a cross reactivity with anti-mouse IL-27 p28 antibody. This is the first report of cloning and characterization of IL-27 p28 in pig.     

IFN-α对山羊副流感病毒3型的抗病毒活性

旨在探讨原核表达纯化的山羊α干扰素(IFN-α)对山羊副流感病毒3型(CPIV3)的抗病毒活性。通过分析山羊IFN-α的序列特点,比对不同种属IFN-α的同源性,进而构建山羊IFN-α成熟蛋白编码基因(去除信号肽基因序列)的原核表达载体pET-30a-gIFN-α,将其转化至感受态细胞Rosetta (DE3),IPTG诱导后镍柱及亲和纯化获得山羊IFN-α。利用RT-qPCR测定山羊IFN-α作用于牛肾细胞(Madin-Darby bovine kidney cell, MDBK cell)后6种干扰素刺激基因(interferon-stimulated genes, ISGs)的相对表达水平;同时,利用TCID₅₀及Western blot测定其对CPIV3的抗病毒活性。结果表明,原核表达的山羊IFN-α蛋白含量为0.20 mg·mL^-1,Western blot结果表明表达产物相对分子质量约为20 ku,与预期结果相符。RT-qPCR结果显示,山羊IFN-α孵育MDBK细胞后,可显著刺激RSAD2、STAT1及ISG15等6种ISGs基...     

A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.     

Identification of canine alpha-lactalbumin

The nucleotide sequence of canine alpha-lactalbumin cDNA from canine mammary tissue was determined by polymerase chain reaction with degenerate primers. A 742 base pairs nucleotide sequence cloned was similar to the size of mRNA in Northern blot analysis. The cDNA encodes 142 amino acid residues containing the conserved sequence motif of alpha-lactalbumin, demonstrating the highest homology with pig (73% identity-82% similarity) among the known amino acid sequences of alpha-lactalbumin. The canine cDNA also showed 71% identity-78% similarity with human, 58-73% with mouse, 60-74% with rat, 67-77% with goat, 66-77% with cattle, and 67-76% with sheep, respectively.     

奶山羊IL-22的原核表达

根据signal IP3.0信号肽分析软件分析结果,设计IL-22不含信号肽表达引物,PCR扩增获得奶山羊IL-22基因,连接表达载体pET-32a,重组质粒转化BL21感受态细胞。对阳性克隆进行诱导表达,对诱导温度、IPTG诱导浓度、诱导时间等条件进行优化,在最佳条件大量诱导蛋白表达。结果显示,在37℃、IPTG浓度为0.1mmol/L、诱导5 h可获得IL-22最大表达量,目的蛋白以包涵体形式存在,包涵体经洗涤、变性和复性,纯度可达90%以上。本研究为进一步探讨IL-22在奶山羊乳腺免疫中的作用奠定了基础。     

Molecular Cloning and Expression of IL2 cDNA from the Tibet Pig

Interleukin-2 is a vital cytokine secreted by activated T lymphocytes, and plays important role in the regulation of cellular and humoral immunity of animals. In our experiment, IL2 cDNA of the Tibet Pig was first cloned by RT-PCR from ConA-stimulated lymphocytes in the blood and subcloned into pMD-18 T vector, which then was identified with endonuclease restriction. The sequencing result showed that Tibet pig IL-2 (TPIL-2) cDNA was 503 bp long (ORF was 465 bp) (Genbank accession number: AY 294018). The recombinant prokaryotic and eukaryotic expression plasmids of the cDNA were then constructed to analyse the ability to stimulate the proliferation of porcine lymphocytes in vitro. The recombinant porcine IL-2 expressed in the prokaryotic cells was found to be of 43 kDa molecular mass, which was consistent with a 17.4 kDa protein deduced from the IL-2 cDNA sequence (glutathione S-transferase molecular mass is 26 kDa); the recombinant protein in eukaryotic cells was confirmed by use of specific rabbit anti-porcine IL-2 serum in an ELISA. The bioactivity of TPIL-2 was detected through MTT colorimetry by stimulating the proliferation of pig ConA-stimulated blasts in vitro. The results indicate that the TPIL-2 significantly promoted the proliferation of ConA-stimulated blasts of pig. This confirms that IL-2 cDNA of the Tibet pig was successfully cloned and expressed in prokaryotic and eukaryotic cells, which lays the foundation for the the preparation of specific recombinant IL-2 protein and development of novel immune adjuvants to raise the immunity of pigs against various infectious pathogens and increase the immunoprotective efficacy of vaccines.     

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.