猪IL-2与IL-6的融合表达及其产物的促淋巴细胞增殖活性

为了获得具有猪白细胞介素-2(pIL-2)和猪白细胞介素-6(pIL-6)双重活性的融合蛋白,研究其作为高效免疫佐剂的可行性,作者利用基因重组技术将克隆到的pIL-2和pIL-6基因的成熟肽片段利用一段柔性Linker序列串联,然后插入到原核表达载体pBV220的适当位置,获得重组质粒pBVpIL-6-2,转化E.coli DH5α、E.coli BL21(DE3)和E.coli Rosetta(DE3)后,42℃诱导表达得到相对分子质量约为36.7ku的重组蛋白pIL-6-2,对蛋白进行纯化、复性后用MTT法检测其对小鼠脾淋巴细胞的促增殖活性,结果显示不同浓度的pIL-6-2蛋白对小鼠脾淋巴细胞的促增殖活性差异很大,其中以0.1μg·mL-1浓度的pIL-6-2活性最好。本研究为利用该蛋白作为高效免疫制剂的应用奠定了良好的基础。     

猪IL-2和IL-6的原核表达及多克隆抗体的制备

IL-2和IL-6是机体重要的免疫调节因子,在佐剂的应用方面具有很好的发展前景。论文以pTIL-2和pTIL-6为模板进行PCR扩增得到猪白细胞介素2(pIL-2)和猪白细胞介素6(pIL-6)的全基因序列,通过EcoRⅠ和HindⅢ双酶切及连接反应将目的基因亚克隆到原核表达载体pET30a中构建了融合表达质粒pETIL-2和pETIL-6。将重组质粒分别转化大肠埃希菌BL21(DE3)和Rosetta(DE3),37℃在IPTG诱导下高效表达了pIL-2和pIL-6,分子质量分别约为26ku和30ku。将包涵体纯化并复性后免疫家兔,制备的兔抗pIL-2和pIL-6的多克隆抗体经ELISA检测效价在1∶12800以上。     

猪白细胞介素-2在甲醇酵母中的表达

将猪白细胞介素-2(pIL-2)的完整cDNA序列克隆到甲醇酵母(Pichiapastoris)表达载体pPIC3 5K中,在E.coli体系中鉴定得到阳性克隆子pPIC3 5K-IL2。将pPIC3 5K-IL2经SacI线性化后,转化入经LiC1致敏的P.PastorisGS115菌株中,得到的重组菌株经1%浓度甲醇诱导后,利用SDS-PAGE电泳,斑点杂交及去糖基化酶消化证实,在培养上清中获得了分泌型表达的猪白细胞介素-2(蛋白分子量约为20KD),其表达量可达到80mg/L。用CTLL-2细胞进行活性检测,生物学活性可达2×106IU/ml。对其表达情况进行时间梯度分析,确定第5天表达量达到最高点,为最佳诱导时间;分析连续4个批次的重组菌株表达情况,结果表明重组菌株在适当菌体浓度下连续培养均能稳定、高效的表达外源蛋白pIL-2。本实验为进一步大规模生产pIL-2提供了理论依据。     

猪白细胞介素6基因的克隆及其作为核酸免疫佐剂的研究

为研究猪白细胞介素6 (pIL-6)作为核酸免疫佐剂的可行性,本研究采用RT-PCR方法,从猪外周血淋巴细胞中扩增出639 bp的pIL-6基因,编码212个氨基酸残基.将pIL-6基因克隆至真核表达载体pVAX构建重组质粒pVAX-IL6,并转染于COS-7细胞后,通过western blot检测到pIL-6在细胞中的表达.将含有猪传染性胃肠炎病毒(TGEV)S基因的重组真核表达质粒pVAX-S与pVAX-IL6混合后腿部肌肉注射6周龄NIH小鼠,通过间接ELISA以及流式细胞仪分别检测免疫小鼠血清中特异性TGEV IgG抗体和外周血T淋巴细胞亚群数量,结果表明pVAX-S+pVAX-IL6免疫组的特异性抗体水平与外周血T淋巴细胞亚群数量显著高于pVAX-S+-pVAX对照免疫组(p＜0.05),显示了pIL-6作为核酸免疫佐剂的良好应用前景.     

猪白细胞介素15基因的克隆及在大肠杆菌中的表达

本研究通过RT-PCR方法从用ConA刺激的猪外周血淋巴细胞中扩增出猪IL-15(pIL-15)编码序列的cD-NA,将其克隆至T载体pMD18-T后,序列测定表明pIL-15基因长度为489 bp,编码162个氨基酸。进一步通过PCR方法获得缺失其N端48 aa的信号肽序列的成熟pIL-15蛋白基因,将其亚克隆到原核表达载体pET-28a的6×His下游,构建重组表达质粒pET-pIL15,转化大肠杆菌BL21(DE3),IPTG诱导,重组菌体裂解物经SDS-PAGE可检测到分子量约为15 ku的重组蛋白,其表达量占总菌体蛋白量的17.5%。Western blot试验也证实表达的重组融合蛋白6×His-pIL15能够很好地与抗6×His单抗发生反应。猪IL-15基因的克隆、表达为进一步研究其功能和应用奠定了基础。     

荣昌猪白细胞介素-2基因的原核表达

应用DNA重组技术,将克隆的荣昌猪白细胞介素-2基因亚克隆到PET-32a( )表达载体上,构建重组表达载体。酶切鉴定正确的重组质粒转化大肠杆菌BL21,用1 mM的HPTG诱导表达重组蛋白。RT-PCR方法检测表明白细胞介素-2基因得到了转录;对表达蛋白进行SDS-PAGE电泳,发现在相对分子质量约37 Ku处有明显的蛋白质条带,而对照组无相应条带产生。最后用MTT法检测表达蛋白的生物学活性,表明所获得的重组蛋白具有较好的生物学活性,这为利用该蛋白及其基因研制高效抗病免疫制剂和基因工程疫苗奠定了良好的基础。     

猪IL-18在酵母中表达及活性检测

以pGEMT/pIL-18为模板,应用PCR法扩增出猪IL-18基因,用EcoR Ⅰ和Xba Ⅰ双酶切后,插入酵母表达载体pPICZαA中,经酶切、PCR扩增及序列测定,成功构建了酵母重组表达载体pPICZ/pIL-18,电击转化毕赤酵母X-33,应用Zeocin筛选获得高拷贝重组转化菌株,甲醇诱导表达,经SDS-PAGE电泳分析重组pIL-18蛋白的表达,并用SephadexG200纯化表达的重组pIL-18蛋白.运用MTT法检测其生物学活性.试验结果表明,重组X-33酵母菌株能够表达分泌性pIL-18,诱导72 h表达量最高,其表达量占总蛋白表达量的38％.而且纯化的重组pIL-18蛋白具有明显促进淋巴细胞增殖的活性.     

猪白细胞介素-2的原核表达及其生物学活性研究

本研究采用RT-PCR方法自刀豆素（Con A）刺激的猪外周血淋巴细胞总RNA中扩增克隆了猪白细胞介素-2（porcine interleutin-2,PoIL-2）成熟肽基因,并亚克隆入pQE30载体进行原核表达,对表达的融合重组猪白细胞介素-2（rPoIL-2）蛋白通过尿素变性、低浓度复性液复性、PBS溶液透析等步骤进行纯化,采用MTT法测定rPoIL-2蛋白促小鼠细胞毒性淋巴样系CTLL-2株细胞增殖活性以评价rPoIL-2的活性。结果表明,成功克隆了rPoIL-2的成熟肽基因,基因全长405 bp,编码134个氨基酸;表达的rPoIFN-α蛋白分子质量大小约为16.5 ku,与预期大小相一致;经纯化后的蛋白质纯度在96%以上;纯化的rPoIL-2蛋白促小鼠CTLL-2株细胞增殖活性最高值是对照细胞的3倍。本研究成功表达了具有生物学活性的PoIL-2蛋白,为新型基因工程抗病毒制剂和免疫增强剂的开发奠定了基础。     

猪白细胞介素18成熟蛋白基因的克隆及在大肠杆菌中的表达

通过RT—PCR方法从用PHA和LPS刺激的猪脾脏细胞中扩增出猪白细胞介素18(pIL-18)成熟蛋白基因的cDNA，克隆到T载体pUCm-T后，序列测定表明，pIL-18成熟蛋白基因核苷酸长度为474bp，编码157个氨基酸。将该基因片段克隆到大肠杆菌表达载体pET-28a构建重组质粒pETIL18m，转化大肠杆菌BL21(DE3)，并用IPTG诱导。重组菌菌体裂解物SDS—PAGE可检测到相对分子质量为19000的重组蛋白，免疫印迹法证实该重组蛋白可以与人IL-18单抗发生特异性免疫反应。     

猪白细胞介素-2在BHK-21细胞中表达及其活性分析

为鉴定BHK-21细胞中稳定表达猪白细胞介素-2(PoIL-2)的活性,本研究利用RT-PCR技术从猪的淋巴细胞中克隆PoIL-2基因,将其连接于pcDNA3.1载体中,并转染BHK-21细胞.经过G418筛选及RT-PCR鉴定,获得稳定表达PoIL-2基因的BHK-21细胞系.采用实时定量PCR及ELISA测定PoIL-2在BHK-21细胞内的表达,同时以淋巴细胞增殖试验(MTT)检测PoIL-2蛋白活性.试验结果表明稳定整合PoIL-2基因的BHK-21细胞系其PoIL-2 mRNA的转录效率是阴性对照组1.43倍,蛋白表达量是阴性对照组1.29倍.MTT试验表明真核表达的PoIL-2促淋巴细胞增殖活性与阴性对照组相比差异极显著(p＜0.01),表明BHK-21细胞表达的PoIL-2具有良好的生物学活性.本研究为开发新型基因工程疫苗佐剂和抗病毒制剂奠定了基础.     

Expressed gene sequences of the equine cytokines interleukin-17 and interleukin-23

This report describes the initial cloning and characterization of the equine interleukin-17 (IL-17) expressed gene sequence from mRNA obtained from equine intestinal tissue and interleukin-23 (IL-23) expressed gene sequence from mRNA obtained from equine peripheral blood mononuclear cells. Equine IL-17 has 462 nucleotides in the translated region, determined by homology with known human and mouse sequences, and shares 84% and 75% identity, respectively. For the deduced amino acid sequences, the identity with human and mouse is 76% and 70%. Equine IL-23 has 579 nucleotides in the translated region. Homology with known human and mouse sequences was determined to be 89% and 77%. Deduced amino acid identities are 89% with the human sequence and 70% with the mouse sequence. The gene sequences were identified as part of the U.S. Veterinary Immune Reagent Network with a goal of developing reagents in order to aid veterinary researchers in the investigation of diseases in livestock species.     

Local interleukin-2 and interleukin-12 therapy of bovine ocular squamous cell carcinomas

Interleukin-2 and interleukin-12 have been used independently to successfully treat the induced and the spontaneous tumours in animals. This trial was done to determine if a combination of IL-2 and IL-12 in the treatment of spontaneous bovine ocular squamous cell carcinomas (BOSCC) would be more successful than IL-2 or IL-12 therapy by themselves. For this trial, we selected 25 BOSCC tumours seen on Holstein Fresian cows in Beatrice, Zimbabwe. The cows were randomly assigned to a treatment group of 5 days of IL-2 (200,000 U/day), 5 days of IL-12 (0.5 microg/day) or 5 days of IL-2 (200,000 U/day) and IL-12 (0.5 microg/day). At 20 months after treatment, the IL-2 therapy group had 63% complete regressions; the combination group had 38% complete regressions, which were significantly higher than the IL-12 group, which had 0% complete regressions at 20 months, despite having 29% complete regressions at 6 months. These results show that IL-2 therapy by itself and in combination with IL-12 is more successful than IL-12 by itself. However, combination therapy does not improve the outcome in comparison to IL-2 as a single therapy. It also proves that IL-2 is consistently successful in the therapy of BOSCC with over 60% complete regression, which corresponds to a number of other studies we have done on IL-2 therapy of BOSCC [Rutten, V.P.M.G., Klein, W.R., De Jong, W.A., Misdorp, W., Den Otter, W., Steerenberg, P.A., De Jong, W.H., Ruitenberg, E.J., 1989. Local interleukin-2 therapy in bovine ocular squamous cell carcinoma. A pilot study. Cancer Immunol. Immunother. 30, 165--169; Stewart, R.J.E., Hill, F.W.G., Masztalerz, A., Jacobs, J.J.L., Koten, J.W., Den Otter, W., 2003. Local low dose interleukin-2 therapy of bovine ocular squamous cell carcinomas in cattle in Zimbabwe, submitted for publication; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H.M., Rutten, V.P.M.G., Ruitenberg, E.J., 1993. Low doses of interleukin-2 can cure large bovine ocular squamous cell carcinoma. Anticancer Res. 13, 2453-2455; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H., Rhode, C., Stewart, R., Faber, J.A., Ruitenberg, E.J., 1995. Therapy of bovine ocular squamous cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up. Cancer Immunol. Immunother. 41, 10-14].     

山羊IL-2的体外表达及其多克隆抗血清的制备

应用RT-PCR方法从山羊外周血淋巴细胞中克隆了山羊白细胞介素-2(Caprine interleukin-2,CapIL-2)基因并进行测序,将编码山羊IL-2蛋白的基因亚克隆至pET30a( )上,转化大肠杆菌BL21(DE3)并用IPTG于37℃诱导培养。表达融合蛋白通过ProBondTM蛋白纯化试剂盒纯化。纯化产物经3次免疫新西兰白兔,制备出山羊IL-2抗血清。本次试验成功地表达、纯化了山羊IL-2,并制备出兔抗山羊IL-2抗血清,为下一步开展山羊IL-2重组蛋白的应用研究奠定了基础。     

Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis

Abstract The experimental hypothesis testéd was that dogs with generalized demodicosis have significantly lower levels of interleukin-2 (IL-2) production and IL-2 receptor expression as compared with normal dogs. The specific objective of this study was to compare the production of IL-2 and IL-2 receptor expression of peripheral blood mononuclear cells in normal dogs and dogs with generalized demodicosis. Ten dogs with juvenile-onset generalized demodicosis were evaluated. Dogs with generalized demodicosis had a significantly lower in vitro lymphocyte blastogenesis response (P < 0.006), fewer cells expressing IL-2 receptors (P < 0.03), and decreased IL-2 production (P < 0.01) than control dogs. Resumen La hipótesis experimental fue que los perros con demodicosis generalizada tienen niveles significativamente inferiores de producción y expresión de receptores de interleuquina-2 (IL-2) comparado con perros normales. El objetivo especifico de este estudio fue el de comparar le producción y expresión de receptores de IL-2 en células mononucleares de sangre periférica en perros normales y en perros con demodicosis generalizada. Se evaluaron diez perros con demodicosis generalizada iniciada en edad temprana. Los perros con demodicosis generalizada tenian in vitro una respuesta blastogénica linfocitaria inferior (P < 0.006), menos células con expresión de receptores de IL-2 (P < 0.03) y una disminución en la producción de IL-2 (p<0.01) respecto a los perros control. [Lemarie, S.L., Horohov, D.W. Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis. (Evaluación de la producción y expresión de recepetores de interleuquina-2 en perros con demodicosis generalizada.) Veterinary Dermatology 1996; 7 : 213–219.] Résumé L'hypothèse expérimentale est que les chiens à démodécie généralisée présente une diminution significative du taux d'interleukine 2 (IL-2) et de l'expression des récepteurs de l'interleukine 2 comparativement à des chiens sains. L'objectif de cette étude est de comparer la production d'I12 et l'expression des récepteurs de l'IL-2 par des lymphocytes sanguins périphériques de chiens témoins et de chiens atteints de démodécie généralisée. Dix chiens avec une démodécie généralisée juvenile ont été testés. Les chiens à démodécie généralisée présentent une transformation lymphoblastique plus basse (p< 0.006), moins de cellules exprimant les récepteurs IL-2 (p<0.03) et une diminution de la production d'IL-2 (p<0.01) par rapport aux chiens témoins. [Lemarie, S.L., Horohov, D.W. Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis. (Evaluation de la production d'interleukine 2 et de l'expression des récepteurs de l'interleukine 2 chez des chiens à démodécie généralisée.) Veterinary Dermatology 1996; 7 : 213–219.] Zusammenfassung Es wurde die experimentelle Hypothese getestét, daß Hunde mit generalisierter Demodikose über signifikant niedrigere Spiegel an Interieukin2-(IL-2)Produktion und IL-2-Rezeptorexpression verfügen im Vergleich zu gesunden Hunden. Der wesentilche Gegenstand dieser Untersuchung war, die Produktion von IL-2 und die IL-2Rezeptorexpression in den mononukleären Zellen des peripheren Blutes bei gesunden Hunden und solchen mit generalisierter Demodikose zu vergleichen. Zehn Hunde mit juvenilem Beginn von generalisierter Demodikose wurden untersucht. Hunde mit generalisierter Demodikose hatten eine signifikant niedrigere in vitro-Reaktion der Lymphozytenblasogenese (P < 0, 006), weniger Zellen, die IL-2Rezeptoren zeigten (P < 0,03) und eine verminderte IL-2Produktion (P < 0,01) als die Kontrollhunde. [Lemarie, S.L., Horohov, D.W. Evaluation of interleukin-2 production and interleukin-2 receptor expression in dogs with generalized demodicosis (Untersuchungen über die Interleukin2-Produktion und Interleukin2-Rezeptorexpression bei Hunden mit generalisierter Demodikose) Veterinary Dermatology 1996; 7 : 213–219.]     

Brain interleukin-1 is involved in blood interleukin-6 response to immobilization stress in rats

Interleukin (IL)-6, a cytokine for host defense responses to infection and inflammation, is known to be induced by non-invasive physical or psychological stress, too. To test possible involvement of brain IL-1 in the stress-induced IL-6 production, IL-1 mRNA expression in the hypothalamus, in parallel with blood IL-6 level, was examined in rats subjected to restriction of their movement (immobilization stress). When rats were immobilized, the hypothalamic IL-1 beta mRNA level was increased in 1 hr, followed by progressive rises in the serum IL-6 level. The immobilization-induced rise in serum IL-6 was mimicked by intracerebroventricular (icv) administration of IL-1 beta under normal conditions, whereas it was attenuated by icv injection of an IL-1 receptor antagonist. These results indicate that IL-1 in the hypothalamus plays a pivotal mediating role in the stress-induced peripheral IL-6 production.     

Enhancement of the interferon gamma assay to detect paratuberculosis using interleukin-7 and interleukin-12 potentiation

A limitation to the widespread application of interferon gamma (IFN-γ) response assays has been logistical difficulties, as they must be performed within hours of blood collection. Detection of specific IFN-γ responses to Mycobacterium avium subspecies paratuberculosis (MAP) as part of the cell-mediated immune response of ruminants with Johne's disease could aid in diagnosis and control programs. In this study, a modified protocol was developed in which cultures were supplemented with interleukin (IL)-7, a survival factor required to maintain resting T cells, alone or in combination with IL-12 to potentiate IFN-γ responses and extend blood storage time. The combination of IL-7 and IL-12 was synergistic, giving IFN-γ responses greater than with IL-12 alone, for sheep blood stored up to 2 days. In a cohort of naturally infected sheep, the same number of animals was identified as test positive using the modified assay (performed after 2 days with IL-7/IL-12 supplementation) as the standard IFN-γ assay performed on the day of blood collection. The modified assay offered greatest advantage in the detection of early stages of paratuberculosis infection, for sheep with low grade and paucibacillary lesions, and at early time points post-infection. The potentiation protocol allowed for practical shipment of blood samples from farm to laboratory, extending permissible transit times and applicability of IFN-γ testing to detect Johne's disease.     

家禽白细胞介素2的研究进展

白细胞介素2（Interleukin-2，IL-2）是一种T细胞生长因子，它还兼有很多免疫调节功能和其他生物学活性。家禽IL-2的结构与哺乳类动物IL-2的结构不同，其功能也有一定差异。目前对于家禽IL-2的认识还远落后于哺乳类动物IL-2。     

白细胞介素18研究进展

白细胞介素 1 8是一种能诱导IFN γ产生的新型细胞因子。它具有多种生物学功能 ,能促进T细胞增殖 ,增强NK细胞的细胞毒作用 ,在诱导Th1细胞的分化成熟过程和Th1细胞为主的细胞免疫反应中具有促进和调节作用。在疫苗佐剂、多肽性药物的开发等方面有着潜在的应用前景     

The characterisation of equine interleukin-1

Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E. coli lipopolysaccharide. Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD. Chromatofocusing of the 17-18 kD peak identified four active fractions. Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9. The pI 7 molecule is probably the equine form of IL-1 beta.