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1.
Funke C King DP McBain JF Adelung D Stott JL 《Veterinary immunology and immunopathology》2003,93(1-2):69-79
Interleukin-6 (IL-6) is a cytokine that can reach detectable systemic levels and is a major inducer of the acute phase response. As such, clinical assays to identify this cytokine in mammalian sera are of diagnostic value. A 558 base-pair (bp) fragment of killer whale IL-6 was cloned and expressed as a 21 kDa protein in Escherichia coli. Biological activity of the recombinant killer whale IL-6 (rkwIL-6) was demonstrated using the IL-6-dependent B9 mouse hybridoma cell line; acute phase sera from a killer whale and supernatants from lipopolysaccharide (LPS)-stimulated killer whale peripheral blood mononuclear cells (PBMCs) also supported the proliferation of the B9 hybridoma. Rat anti-mouse IL-6 receptor antibody effectively blocked biological activity of all three sources of IL-6. Polyclonal antisera, specific for the recombinant protein, were obtained by successive immunization of a rabbit with rkwIL-6. The polyclonal antibody was capable of neutralizing the biological activity of both recombinant and native kwIL-6. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal rabbit anti-rkwIL-6 and the recombinant protein; sensitivity of the assay was in the range of 1 ng/ml. The ELISA was subsequently used to identify the presence of native IL-6 in acute phase sera of two species of delphinidae, a killer whale and a bottlenose dolphin. The application of quantitative cytokine assays as diagnostic tools for monitoring cetacean health are becoming feasible as many animals are now being trained for fluke presentation, making blood collection a routine procedure. 相似文献
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FAN Si-ning ZHANG Hai-ling ZHAO Jian-jun WANG Yang HU Bo LV Shuang MA Fan-shu LI Xin-tong LING Ming-yu ZHAO Hui LIAN Shi-zhen YAN Xi-jun 《中国畜牧兽医》2017,44(1):30-37
In order to obtain high purity of interleukin 4 (IL-4) protein,the specific primers were designed and synthesized according to the mink IL-4 gene sequence in GenBank. The IL-4 gene was amplified by RT-PCR using total RNA of the lymphocyte isolated from the peripheral blood that induced by phytohemagglutinin (PHA). The length of IL-4 gene sequence was 399 bp which encoded 132 amino acids. Phylogenetic analysis revealed that the amino acid sequences homology between mink and ferret was 99.2%,90.0% with Ailuropoda melanoleuca and Canis familiaris. Prokaryotic expression vector pProEX-HTb-IL-4 was constructed and induced by IPTG. The recombinant protein of 15 ku was isolated by SDS-PAGE and detected by Western blotting. Highly purified recombinant protein of mink IL-4 was obtained by His-Trap HP affinity columns method. This research laid the foundation for the further studies on the biological function of mink IL-4 gene. 相似文献
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为了获得高纯度的白细胞介素-4（IL-4）蛋白,对分离得到的水貂外周血淋巴细胞经植物血凝素（PHA）诱导后,提取淋巴细胞总RNA,根据GenBank中登录的雪貂IL-4基因序列,设计并合成特异性引物,通过RT-PCR扩增获得了水貂IL-4基因序列全长399 bp,编码132个氨基酸,与雪貂氨基酸序列同源性高达99.2%,与熊猫、犬同源性均为90.0%。将编码成熟蛋白基因构建到原核表达载体pProEX-HTb,IPTG诱导后,SDS-PAGE及Western blotting结果显示,IL-4表达产物为15 ku的包涵体蛋白。通过His-Trap HP亲和层析预装柱变性、复性洗脱可获得高纯度的重组IL-4蛋白。本试验为水貂IL-4基因的进一步研究奠定了基础。 相似文献
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根据GenBank发表的牛白细胞介素2(BoIL-2)基因序列,设计1对特异性引物,应用RT-PCR技术扩增出重组牛白细胞介素2(rBoIL-2)基因。将克隆片段与pMD18-T载体连接,并经过酶切及PCR鉴定,测序结果显示,克隆的BoIL-2基因与GenBank发表的BoIL-2基因序列同源性为98.7%。将测序正确的克隆片段与pET30a(+)载体连接,构建重组表达质粒pET30a(+)-rBoIL-2,通过双酶切及PCR鉴定阳性的重组质粒进行序列测定后在大肠杆菌的表达,分子质量约为23.49 ku;表达产物主要分布在包涵体中。Western blotting证实所得到的重组蛋白为BoIL-2重组蛋白。用镍离子亲和树脂对所得的BoIL-2重组蛋白进行纯化,并免疫新西兰兔成功制备兔抗rBoIL-2多克隆抗体,为下阶段的研究提供了重要的试验材料。 相似文献
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将猪白介素4(Porcine interleukin4,PIL-4)基因亚克隆到真核表达载体pEGFP-N1中,构建重组表达质粒pEGFP-PIL-4,利用脂质体法转染CHO-K1细胞,采用荧光显微镜实时观察、RT-PCR和Western-blot分别检测CHO细胞转录表达目的分子的情况,通过MTT法检测所表达蛋白的生物学活性。结果在将重组质粒转染CHO-K1细胞中24、48 h后的均观察到绿色荧光;经G418筛选14~20 d后转染的细胞形成了阳性细胞集落,用RT-PCR扩增出约339 bp的目的基因片段;经Western-blot检测到约为39 000的特异蛋白分子条带,并证明在转染重组质粒的细胞中表达了高生物活性的重组蛋白。 相似文献
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本试验旨在构建表达猪布鲁菌（Brucellasuis）外膜蛋白0mp25蛋白的真核重组表达质粒,并探讨Omp25蛋白对猪肺泡巨噬细胞生长活性及细胞因子分泌的影响。以猪布鲁菌基因组DNA为模板,设计带有6×His标签的引物克隆Orap25基因,扩增产物克隆入pCI-neo真核表达载体,双酶切及测序鉴定正确后转染猪肺泡巨噬细胞3D4／21,检测其对巨噬细胞生长活性和分泌细胞因子的影响。RT—PCR和Westernblotting检测结果证实Omp25蛋白获得正确表达。显微镜观察细胞形态表明,Omp25蛋白对猪肺泡巨噬细胞的形态无明显影响。MTT结果表明,Orap25蛋白对猪肺泡巨噬细胞的生长活性有轻度但不显著的抑制作用。ELISA检测TNF—α和IL-12蛋白表达水平表明,Omp25蛋白可以显著抑制巨噬细胞分泌TNF-α和IL-12。本试验成功构建了表达猪布鲁菌外膜蛋白Omp25蛋白的真核重组表达质粒,该蛋白对猪肺泡巨噬细胞的细胞形态没有明显影响,对其生长活性有轻度但不显著的抑制作用,可以显著抑制巨噬细胞分泌TNF—α和IL-12。 相似文献
7.
为研究杂交猪白细胞介素-18(IL-18)蛋白的结构与功能,将杂交猪外周血淋巴细胞体外刺激培养后,提取淋巴细胞总RNA,通过RT-PCR扩增的方法,获得IL-18全基因,克隆到pMD18-T载体中进行测序,测得核苷酸序列与GenBank登录的参考序列的同源性在99.0%～99.8%之间,推到氨基酸同源性在99.7%～100%之间;然后亚克隆到pET30a(+)表达载体中,构建并筛选出阳性重组子,标记为pET30a/IL-18。将阳性重组质粒转化进RossettaTM宿主菌中,通过改变IPTG浓度、诱导温度和诱导时间,使重组蛋白获得表达,并确定最佳诱导条件为:IPTG终浓度1.0mM、诱导温度37℃、诱导时间为3-4h。经SDS-PAGE和Western-blotting分析表明该重组蛋白相对分子量约为35Ku,表达量约为38%,主要存于包涵体中,具有良好的免疫学活性。 相似文献
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试验旨在研究猪腺病毒3型（PADV3） Protease蛋白在大肠杆菌中的表达,并制备该蛋白的多克隆抗体。利用PCR扩增PADV3 Protease基因,构建重组原核表达载体pET28a-PADV3-Protease和真核表达载体pEGFP-PADV3-Protease,采用双酶切和测序鉴定;将原核表达载体pET28a-PADV3-Protease转化大肠杆菌BL21（DE3）感受态细胞得到重组原核表达菌株,经IPTG诱导收集蛋白,采用SDS-PAGE和Western blotting鉴定,将目的蛋白纯化后与佐剂乳化制备免疫原免疫家兔,制备Protease蛋白多克隆抗体;将真核表达载体pEGFP-PADV3-Protease转染HEK293细胞经G418筛选建立稳定表达EGFP-Protease融合蛋白的细胞系,以该稳定表达细胞系为包被抗原,采用免疫过氧化物酶单层细胞染色法（IPMA）检测抗体的免疫活性与抗体滴度。结果显示,Protease基因开放阅读框（ORF）为615 bp,原核表达系统中Protease蛋白以包涵体形式存在,分子质量大小为23 ku,与真核细胞表达的Protease蛋白分子质量一致,该蛋白在细胞核与细胞质中均有分布,制备的Protease蛋白多克隆抗体能与EGFP-Protease融合蛋白稳定表达真核细胞系发生特异性的反应,与对照细胞无反应。本试验构建了Protease蛋白原核表达菌株和真核表达细胞株,制备的PADV3-Protease蛋白多克隆抗体免疫活性良好,为进一步研究Protease蛋白的生物学功能和PADV3的血清学诊断提供了基础材料。 相似文献
11.
Expression of porcine interleukin-2 in Escherichia coli 总被引：6，自引：0，他引：6
Iwata H Yamamoto M Hasegawa A Kurata K Inoue T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2000,62(10):1101-1104
A mature form of porcine interleukin-2 (IL-2) protein without signal peptides was expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli using pGEX vector. Since most of GST-IL-2 fusion protein was detected in an insoluble fraction on SDS-PAGE analysis, the insoluble fusion protein was solubilized by refolding procedure using urea. The recombinant IL-2 (rIL-2) was purified by a batch method using Glutathione Sepharose 4B and factor Xa digestion and used for preparation of antisera in mice. The antisera reacted with rIL-2 expressed in baculovirus system on immunoblot analysis. In addition, the purified rIL-2 showed a high biological activity on CTLL-2 proliferative response. 相似文献
12.
为研究日本血吸虫凋亡蛋白抑制因子（inhibitor of apoptosis protein of Schistosoma japonicum,SjIAP）重组蛋白诱导BALB/c小鼠的免疫保护效果,利用PCR技术扩增SjIAP基因,构建重组表达质粒pET-28a（＋）-SjIAP,诱导表达重组SjIAP蛋白,并利用重组蛋白制备兔源多克隆抗体血清。然后,选用SjIAP重组蛋白免疫BALB/c小鼠,利用ELISA检测免疫小鼠血清中的特异性抗体水平,以及免疫小鼠脾脏淋巴细胞在SjIAP重组蛋白刺激后产生的细胞因子水平。强化免疫后,将小鼠进行血吸虫尾蚴攻虫试验,感染38 d,进行剖杀,计算虫体减虫率及肝脏减卵率。Western blot结果表明本研究制备的兔源多抗血清能特异性识别SjIAP重组蛋白。ELISA检测表明免疫SjIAP重组蛋白可诱导较高水平的IgG及IgG亚型（IgG1、IgG2a、IgG2b、IgG3）抗体和IFN-γ、IL-2及IL-4细胞因子。动物试验表明,免疫SjIAP重组蛋白的小鼠与PBS组相比分别获得了31.5%的减虫率和37.2%的肝脏减卵率。免疫SjIAP重组蛋白能诱导小鼠获得一定减虫和减卵保护效果,提示血吸虫凋亡蛋白抑制因子可作为抗血吸虫病的疫苗候选分子。 相似文献
13.
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. 相似文献
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为获得活性良好的兔出血症病毒基因工程抗原,本研究对兔出血症病毒ZB分离株的VP60基因进行了原核表达与初步应用。参照ZB株分离病毒VP60基因序列,设计合成一对特异性引物,PCR扩增长876bp的VP60基因片段。将目的片段定向克隆至pET30a表达载体中,经鉴定正确后,重组质粒转化BL21表达菌,经IPTG诱导后获得了以包涵体形式表达的重组蛋白,重组蛋白纯化后,Western blot检测表明具有良好的抗原性与特异性,以该蛋白作为诊断抗原,初步建立了检测兔瘟病毒抗体的间接ELISA诊断方法。本研究为RHDV分子流行病学调查提供了参考,为VP60蛋白结构与功能研究、RHDV抗体检测试剂盒及新型疫苗的研制奠定基础。 相似文献
16.
We recently cloned the genes encoding chicken IL-15 and IL-15 receptor (R) alpha proteins. In this study, 12 monoclonal antibodies (mAbs) against recombinant chicken IL-15Ralpha were produced and characterized. By enzyme-linked immunosorbent assay (ELISA), all mAbs showed binding specificity for IL-15Ralpha, but not IL-2 or interferon-gamma, and identified a 25.0kDa protein by immunoblot analysis. Flow cytometric analysis revealed negligible expression of IL-15Ralpha on non-activated lymphocytes from the spleen, thymus or bursa, low but detectable expression on macrophages and high expression on concanavalin A-activated spleen lymphoblasts. Established chicken T cell (RP13) and macrophage (HD11) cell lines expressed substantially higher levels of IL-15Ralpha compared with a B cell line (RP9). Two mAbs inhibited IL-15 dependent proliferation of T cells suggesting that the tertiary structure of the protein domain of native IL-15Ralpha that binds to IL-15 is preserved in the recombinant receptor molecule. These mAbs will be useful reagents for further in vitro and in vivo studies of the biological functions of chicken IL-15 and its receptor. 相似文献
17.
Cunningham FM Vandergrifft E Bailey SR Sepulveda MF Goode NT Horohov DW 《Veterinary immunology and immunopathology》2003,95(1-2):63-72
The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein. 相似文献
18.
Min W Lillehoj HS Li G Sohn EJ Miyamoto T 《Veterinary immunology and immunopathology》2002,88(1-2):49-56
The chicken IL-15 gene was recently cloned and shown to encode a polypeptide with T cell growth factor activity similar to IL-2. To further characterize the chemical and biological properties of chicken IL-15, we generated a panel of monoclonal antibodies against bacterially expressed protein and characterized their binding specificities. All antibodies were reactive by ELISA with recombinant IL-15, but not IL-2, and identified a 15kDa recombinant chicken IL-15 by Western blot analysis. Two antibodies inhibited IL-15-induced proliferation of splenic lymphoblast cells. These monoclonal antibodies will be useful for further structural and immunological studies of chicken IL-15. 相似文献
19.
SIgA是评价黏膜免疫的重要指标,分泌片(SC)是SIgA的特有成分,为了更方便的获得大量的SIgA的分泌片,本研究应用RT-PCR方法扩增出SC基因片段,将SC基因片段克隆到pGEX-4T-1原核表达载体上,经酶切和测序验证获得pGEX-4T-1-SC重组质粒;将获得的pGEX-4T-1-SC转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导表达重组蛋白rSC.重组蛋白rSC经鉴定和纯化后,按常规免疫程序制备多克隆抗体;利用Western blot对抗重组蛋白rSC的多克隆抗体进行鉴定.结果显示,该抗体能特异性识别猪初乳中的SIgA免疫球蛋白,而与猪初乳中的IgG不发生反应.因此,抗SC重组蛋白的多克隆抗体可以作为抗SIgA免疫球蛋白的特异性抗体,为进一步建立SIgA的检测方法及黏膜免疫的研究奠定基础. 相似文献
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应用RT-PCR技术从新城疫I系疫苗诱导的洛阳土种鸡胚脾淋巴细胞中扩增到全长0.43kb的鸡IL-2基因cDNA,将IL-2cDNA克隆到PMD-T载体,测序结果表明,该基因为不含终止密码子的目的基因。将该目的基因克隆到原核表达载体PET-28a,获得的重组质粒PET-28a-IL2经酶切、PCR及序列测定,表明IL-2基因插入的位点、大小与读码框均正确。PE-28a-IL2在大肠杆菌BL21（DE3）中经IPTG诱导,表达产物经SDS-PAGE电泳分析,在14ku处出现一条约占总蛋白21％的蛋白带,表明所克隆的IL2基因在大肠杆菌中得到良好的表达,为进一步研究IL-2的生物学特性奠定了基础。 相似文献