Molecular Cloning, Expression, and In Silico Structural Analysis of Guinea Pig IL-17

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.     

豚鼠生长激素受体cDNA的克隆

报道了豚鼠肝生长激素受体（ＧＨＲ）的ｃＲＮＡ克隆和编码区序列。它由１８９９ｂｐ组成,编码６１０个氨基酸。此外,还报道豚鼠ＧＨＲ的结构特征和同源性比较的结果。     

猪电压依赖性阴离子通道1基因的分离及物理定位

从猪胚胎骨骼肌cDNA文库中筛选出一克隆子,通过测序及电子延伸获得包含全长CDS的猪VDAC1基因cDNA序列。比对发现此基因在核苷酸和氨基酸水平与人及小鼠都具有较高的同源性。应用辐射杂种板(RH)对此基因进行染色体的精确定位,定位结果显示VDAC1基因定位在猪2号染色体长臂。     

Strain differences in guinea pigs' bronchial sensitivity to acetylcholine

The bronchial sensitivity to acetylcholine (ACh) of guinea pigs of various strains was investigated to clarify strain differences. Inbred Strain 2, Strain 13 and JY-1 and non-inbred Hartley strain (two colonies) were used in this experiment. (1) Guinea pigs were exposed to 0.08% ACh aerosol and the time needed to produce falling down (TNPFD) was determined. Mean +/- standard error of TNPFD (n = 14 per group) of animals was 182 +/- 28 sec, 148 +/- 22 sec, 210 +/- 30 sec, 342 +/- 24 sec and 406 +/- 36 sec in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. There was a significant difference in TNPFD between inbred strains and non-inbred strains (P less than 0.05 or P less than 0.01), indicating that inbred strains had higher sensitivity. (2) Guinea pigs were exposed to 20-5000 micrograms/ml ACh for 2 min. The mean dose threshold as determined by transcutaneous oxygen pressure was 524 micrograms/ml, 424 micrograms/ml, 614 micrograms/ml, 1317 micrograms/ml and 1651 micrograms/ml (n = 14 per group) in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. Inbred strains showed lower dose thresholds than non-inbred strains. (3) Isolated trachea-lungs of 5 guinea pigs were perfused with 10(-9)-10(-5) g/ml ACh to determine strain differences. Dose response curves of animals of inbred strains shifted to the left (lower concentrations), unlike those of non-inbred strains, suggesting that inbred strains had higher sensitivity to ACh than non-inbred strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

cDNA cloning and nucleotide sequence of lipocortin-like 33 kDa protein in guinea pig neutrophils

cDNA clones of guinea pig neutrophil 33 kDa protein, a lipocortin like-protein, were isolated from two lambda gt10 libraries and one primer-extended lambda gt10 library of guinea pig neutrophils using synthetic oligonucleotide probes or cDNA fragment probe. The cDNA consists of 1389 nucleotides, and contains 1038 nucleotides encoding 346 amino acids of 33 kDa protein and a poly(A) tail. The deduced amino acid sequence shows high homology with those of lipocortin 1 from human U937 cells (89% homology) and rat lung (86%).     

Prokaryotic Expression and In Vitro Functional Analysis of IL-1β and MCP-1 from Guinea Pig

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.     

Antigen-induced suppression of mitogen responses and resistance to experimental autoimmune encephalomyelitis

Random-bred Hartley and inbred Strain 2 and Strain 13 guinea pigs were inoculated for acute experimental autoimmune encephalomyelitis (EAE). Sixty-six percent (69/103) of the Hartleys developed signs of EAE while the remaining 34% (34/103) were resistant. No Strain 2 and all Strain 13 guinea pigs developed EAE. Histologic examination of nervous tissue revealed that susceptible Hartleys and Strain 13 and Strain 2 animals had lesions characteristic of EAE. Tissue from resistant Hartleys showed fewer and less severe changes. Lymphocyte-transformation assays with EAE-inducing and noninducing antigens and T-cell mitogens revealed three different sets of responses in vitro: (i) lymphocytes from all animals responded to mitogens; (ii) lymphocytes from susceptible animals responded to EAE-inducing antigens; and (iii) lymphocytes from resistant Hartleys were suppressed from responding to the mitogens solely by EAE-inducing antigens. Plasmas from all EAE-sensitized animals had equivalent anti-myelin basic proteins (MBP) antibody titers and skin tests of EAE-inoculated Hartleys were all positive for MBP sensitization. Therefore, resistance and reduced histologic changes characteristic of EAE correlated with a disease-specific antigen-induced suppression of lymphocyte responses to T-cell mitogens. This suggests that clinical resistance to EAE in Hartley guinea pigs is mediated by an immunologic suppressor mechanism.     

Induction of allergic reactions in guinea pigs with purified house dust mite allergens

To establish a guinea pig model for house dust mite allergy with purified mite allergens, we studied the immune response to two major mite allergens, native Der f 1 (nDer f 1) and recombinant Der f 2 (rDer f 2) and crude mite extract in Hartley guinea pigs. Animals were immunized with either mite extract, nDer f 1 or rDer f 2, four times at 2- to 3-week intervals. Then the guinea pigs were examined as to the status of sensitization to the sensitizing antigen. Intradermal injection of mite antigens to mite extract-, nDer f 1-, and rDer f 2-sensitized animals induced both immediate and late-phase cutaneous reactions. Allergic airway disease was also provoked by the intranasal instillation of rDer f 2 or mite extract. Anti-nDer f 1 and -rDer f 2 IgE as well as anti-mite extract IgE were produced in the sensitized guinea pigs and IgE titer for three mite antigens were comparable. We concluded that immunization of Hartley guinea pigs with nDer f 1 and rDer f 2 achieved sensitization to mite allergens, which was comparable to that obtained by the immunization with mite extract. A mite-allergic model suitable for immunological and pharmacological studies was established from rDer f 2-sensitized guinea pigs.     

Schistosoma mansoni: the IMP4 gene is involved in DNA repair/tolerance after treatment with alkylating agent methyl methane sulfonate

Using a functional complementation strategy, we have isolated a Schistosoma mansoni cDNA that complemented Escherichia coli mutant strains which are defective in the DNA base excision repair pathway. This cDNA partially complemented the MMS-sensitive phenotype of these strains. The sequence of the isolated cDNA was homologous to genes involved in the RNA metabolism pathway, especially ScIMP4 of Saccharomyces cerevisiae. To establish whether the S. mansoni cDNA clone could complement yeast ScIMP4-defective mutants, we constructed a yeast haploid strain that coded for a truncated Imp4p protein. This mutant strain was treated with different DNA damaging agents, but showed only MMS sensitivity. The functional homology between the ScIMP4 gene and the cDNA from S. mansoni was verified by partial complementation of the mutant yeast with the worm's gene. This gene appears to be involved in DNA repair and RNA metabolism in both S. mansoni and S. cerevisiae.     

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.     

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.     

CRRP: a guinea pig protein, identified by sequence homology to human CR1, which contains two short consensus repeat motifs and appears not to be transmembrane or secreted.

cDNA from the C4b-binding site of the human C3b/C4b receptor (CR1) was used to find homologous sequences in the guinea pig. This cDNA identified an 18S mRNA species in guinea pig spleen, but not liver. Probing of a guinea pig spleen cDNA library identified clones with identical 1.5-kb inserts, which also hybridized to mRNA in spleen, but not liver. Sequence analysis of the insert revealed a single long open-reading frame coding for a 20,000 Mr protein consisting of two short consensus repeat motifs homologous to human CR1, and unique sequence at the amino- and carboxy-terminals of the short consensus repeats. This sequence did not encode peptides with features of transmembrane domains or signal peptides. Antibody to this complement receptor-related protein-beta galactosidase fusion protein recognized a 20,000 Mr protein in SDS lysates of guinea pig spleen, lymph node, lymphocytes, neutrophils, and peritoneal macrophages. Immunoprecipitation of human serum by this antibody revealed an 180,000 Mr protein reacting both with the anti-guinea pig protein antibody and with anti-human CR1 antibody. Immunoprecipitation of guinea pig serum revealed no protein reacting with the anti-guinea pig protein antibody. Tissue staining of cultured peritoneal macrophages with this antibody showed intracellular staining, as opposed to membrane staining obtained with anti-guinea pig Ig antibody. The lack of membrane expression was confirmed by surface protein radiolabeling experiments and by fluorescent staining of surface proteins. Thus, we have identified a guinea pig protein with homology to human CR1, which may have an unusual property for this class of proteins in that it appears to be intracellular.     

狗肝脏苯甲二氮茸结合抑制因子的cDNA克隆及序列分析

在研究狗抗吗啡活性肽ＰＰＣ过程中,发现它与牛的ＤＢＩ（ｄｉａｚｅｐａｍｂｉｎｄｉｎｇｉｎｈｉｂｉｔｏｒ）的氨基酸序列有很高的同源性,但尚未见到有关狗的ＤＢＩ的文献报道,为了更好的探讨ＰＰＣ和ＤＢＩ相互间的关系,对狗的ＤＢＩ的ｃＤＮＡ进行了克隆和序列分析。本研究利用大鼠ＤＢＩ的基因片段为探针,从狗肝脏ｃＤＮＡ文库中,筛选得到了一阳性克隆,并进行了全自动和手工测序,得到了ＤＢＩ的全长基因。根据ＥＢＭＬｂａｎｋ序列检索,发现狗的ＤＢＩ核酸序列与牛的同源性为８１％,将其核酸序列翻译成氨基酸序列,进行同源序列比较,结果显示：狗的ＤＢＩ的氨基酸序列与猪、牛、人、酵母ＤＢＩ的同源性分别为８８．５％、８７．４％、８３．９％、４６．５％。研究还发现狗的ＤＢＩ序列与抗吗啡活性肽ＰＰＣＮ端６２个氨基酸只有两个不同,Ｃ端１７个氨基酸序列完全相同。只是ＰＰＣ比ＤＢＩ中间多了２３个氨基酸。     

地塞米松对哮喘豚鼠血清白细胞介素-4 变化及肺组织形态的影响

目的:观察地塞米松对哮喘豚鼠血清白细胞介素-4(IL-4)及肺组织形态的影响,探讨糖皮质激素治疗哮喘的机制。方法:30只健康雄性Hartley系豚鼠随机分为空白对照组,病理模型组,地塞米松组。用卵白蛋白(OVA)复制哮喘豚鼠模型。采用流式细胞术分析CD4+interleukin-4(IL-4)细胞占CD4+T细胞的比例,光镜检查肺组织形态变化。结果:病理模型组外周血中IL-4明显高于空白对照组(P<0.05),地塞米松组外周血中IL-4较病理模型组明显降低(P<0.05),且地塞米松组支气管上皮损伤,粘液腺增生等病理改变较病理模型组明显改善。结论:地塞米松治疗哮喘的作用与抑制Th2的活化,改善支气管上皮损伤,粘液腺增生等病理改变有关。