猪白细胞介素4,6融合基因和CpG基序对猪蓝耳病疫苗免疫的强化效应

为探索新型高效安全的猪蓝耳病疫苗免疫佐剂,本实验用离子交联法制备壳聚糖纳米颗粒包裹猪融合基因PIL-46和CpG基序的真核表达质粒(CNP-(VRIL-46 VR1C)),将CNP-(VRIL-46 VR1C)肌注接种过猪蓝耳病灭活苗的长白川白杂交猪,接种后1、2、4、6和8周采取实验猪静脉血,用Sandwich ELISA检测血清中白细胞介素2(IL-2)、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量,同时检测外周血液免疫细胞数量的变化。结果发现实验猪接种CNP-(VRIL-46 VR1C)后1～8周内,其血清中IL-2、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量较对照组均显著增多(P<0.05),淋巴细胞和单核细胞数量也明显升高(P<0.05)。这些表明接种CNP-(VRIL-46 VR1C)的实验猪的特异体液和细胞免疫水平均显著多于对照组,提示PIL-46基因和CpG基序组合能显著增强猪的特异体液和细胞免疫机能,明显提高其免疫防御力,具有研制为高效安全的分子免疫增强剂的应用前景。     

壳聚糖纳米颗粒包裹猪白细胞介素2和融合白细胞介素4/6基因对猪圆环病毒2型疫苗免疫的强化

目的探讨猪Sus scrofa domesticus白细胞介素2(IL-2)和融合白细胞介素4/6(IL-4/6)基因的共表达对仔猪免疫应答的影响,为进一步研制新型免疫调节剂来加强仔猪抵御断奶后多系统衰竭综合征奠定基础。方法将猪IL-2和IL-4/6融合基因的共表达重组质粒,用壳聚糖材料包裹制成纳米颗粒,记作VRIL-4/6-2-CS。将仔猪分为2组,分别肌肉注射VRIL-4/6-2-CS(实验组)和生理盐水(对照组),2组均同时接种猪圆环病毒2型(PCV-2)疫苗,在接种后的第0、7、14、28天采集血样并分析免疫变化。结果实验组仔猪血清中的IgG2a抗体数量和血液中CD4~+、CD8+~T淋巴细胞数量均显著高于对照组(P0.05);同时,实验组仔猪的IL-2、IL-4、IL-6、TNF-α、TLRs(TLR-2,7)、STATs(STAT-1,2,3)基因表达水平也显著高于对照组(P0.05)。尽管2组之间PCV-2特异性抗体的含量差异无统计学意义,但是实验组仔猪的生长速率显著高于对照组(P0.05)。结论 VRIL-4/6-2-CS能促进仔猪对PCV-2疫苗的免疫应答,是一种安全有效的免疫佐剂。     

猪白细胞介素2与融合白细胞介素4/6基因共表达的免疫效应研究

目的构建猪融合白细胞介素4/6与猪白细胞介素2基因的共表达载体,研究其共表达对小鼠免疫的协同效应。方法以2A自剪接技术首次构建猪融合白细胞介素4/6与白细胞介素2基因的共表达重组质粒,以壳聚糖纳米材料包裹制成纳米颗粒,进行体外转染HEK293细胞,提取总RNA进行RT-PCR分析,最后肌肉注射小鼠进行体内实验并分析。结果成功构建了重组共表达质粒VRIL4/6-2;壳聚糖包裹后转染HEK293细胞,48 h后收集细胞,RT-PCR检测显示目的基因能够在HEK293细胞中高效地转录与表达。小鼠接种实验结果表明,实验组血清中的IgG和IgG2a的含量比对照组显著增加,并且VRIL4/6-2-CS接种组的小鼠体液免疫水平明显增高(P0.05);VRIL4/6-2实验组的CD4+淋巴细胞显著多于其它实验组(P0.05);实验组IL-2、IL-4、IL-6、IL-23基因的表达水平明显高于对照组(P0.05),实验组小鼠外周血白细胞、血小板和血红蛋白的含量均显著高于对照组(P0.05)。结论 VRIL4/6-2共表达质粒能够更好的增强动物体液免疫和细胞免疫机能,可作为提高动物体液免疫和细胞免疫的经济高效安全新型免疫调节剂,具有潜在的重要应用前景。     

CpG壳聚糖纳米颗粒对小鼠乙型肝炎疫苗免疫应答的影响

为探索高效安全的分子免疫增强剂,本实验设计合成含11个CpG基序的寡聚核苷酸(CpG ODN),制备壳聚糖纳米颗粒包裹CpG ODN,研究新型CpG ODN壳聚糖纳米颗粒(CpG-CNP)对人乙型肝炎疫苗的免疫佐剂效应.在肌注乙型肝炎疫苗免疫6周龄昆明小鼠的同时,肌注接种裸CpG ODN 30 pmol/只(A1组)和CpG-CNP 5 pmol/只(A2组),口服CpG-CNP 30 pmol/只(B组),并设生理盐水空白疫苗对照组(C组).在接种后每周采血,Sandwich ELISA检测实验小鼠IL-2、IL-4、IL-6、IgG、IgA和IgM水平以及特异性抗体(HbsAb)含量和免疫细胞数量的变化.结果 发现各实验组小鼠的白细胞介素、血清免疫球蛋白、乙肝特异抗体和外周血免疫细胞均较对照组显著增多(P<0.05);CpG-CNP肌注组小鼠血液的免疫球蛋白、特异性乙肝表面抗体和白细胞介素含量、淋巴细胞和单核细胞数量较A1组和B组显著增加(P<0.05).CpG-CNP口服组与裸CpG肌肉注射组无显著差异(P＞0.05).这些表明CpG-CNP不仅能高效诱导小鼠对乙肝疫苗的体液免疫应答,同时也显著增强其细胞免疫应答;CNP包裹可提高CpG的免疫刺激效应,减少CpG剂量,提示新CpG-CNP肌注或口服可用于HBV的免疫预防.     

共表达PCV2 ORF2和猪IL-18基因的重组猪伪狂犬病毒对小鼠的免疫原性

【目的】研制猪伪狂犬病毒(PRV)和猪圆环病毒2型(PCV2)的二联活疫苗,并用猪IL-18作为免疫佐剂。【方法】将猪IL-18基因插入到质粒p GO中,获得的重组转移质粒p GO18与猪PRV弱毒HB98株DNA共转染ST细胞,并进行空斑筛选和纯化;RT-PCR和Western blot分别从转录和蛋白水平鉴定其表达情况。将重组病毒PGO18和PGO、PRV弱毒株HB98、PCV2灭活商品苗和1640细胞培养基分别免疫6周龄雌性昆明小鼠,4周后二次免疫,二免后4周用PCV2 DF强毒和PRV Min/A强毒接种小鼠。通过ELISA、血清中和试验和流式细胞术及攻毒保护试验评价重组病毒的免疫原性。【结果】获得了重组病毒PGO18,并且可在ST细胞内表达;PGO18可诱导小鼠机体产生PCV2的ELISA和PRV的中和抗体水平,刺激CD3+、CD4+、CD8+T细胞亚群的增殖,且能有效抵抗PCV2和PRV强毒攻击。【结论】IL-18基因可增强重组病毒的免疫效果,使重组病毒具有良好的免疫原性,有望成为防治PCV2和PRV的候选疫苗株。     

以CpG为佐剂的犬细小病毒基因疫苗的初步研究

目的研制犬细小病毒(CPV)基因疫苗。方法以CPV VP2基因为基因免疫的目的基因,以pcDNA3和pcDNAK质粒为基因免疫的载体,以非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)为核心的免疫刺激序列为免疫佐剂,构建重组质粒并免疫BALB/c小鼠和毕格犬。结果经pcDNA3-VP2C1(含1个拷贝CpG基序)基因免疫的BALB/c小鼠能产生抗CPV血凝抑制抗体;对于经CPV灭活苗初次免疫的毕格犬,用pcDNAK-VP2C2(含2个拷贝CpG基序)质粒免疫产生的再次免疫应答优于pcDNA3-VP2C1。结论VP2基因、pcDNAK和犬源CpG可用于CPV基因疫苗的进一步研究。     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。     

共表达O型口蹄疫病毒P1-2A基因和猪白细胞介素18的重组鸡痘病毒的构建

目的:为获得能有效预防O型口蹄疫病毒的重组鸡痘病毒活载体疫苗奠定基础。方法：在O型口蹄疫病毒P1-2A基因上游引入Kozak序列,下游通过Linker与细胞因子IL-18联结,获得P1-2A基因与猪IL-18基因融合表达基因盒P1-2A-IL-18,将该表达基因盒克隆至鸡痘病毒中间转移载体pUTAL-3C中,构建重组鸡痘病毒转移载体质粒pUTAL-3C- P1-2A-IL-18。通过脂质体转染法,将pUTAL-3C- P1-2A-IL-18与鸡痘病毒282E4株共转染鸡胚成纤维细胞（CEF）,通过BrdU三次加压筛选,挑选出单克隆重组病毒株。结果：经RT-PCR和间接免疫荧光法鉴定,证明所筛选的1株重组鸡痘病毒在CEF中能正确表达P1-2A-IL-18基因盒。结论：成功获得了一株共表达O型口蹄疫病毒P1-2A基因和猪白细胞介素18基因的重组鸡痘毒疫苗候选株rFPV-3C-P1-2A-IL-18。     

表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌对小鼠的免疫原性

【目的】构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,对重组菌株的生物学特性以及对小鼠的免疫原性进行研究。【方法】分别将猪肺炎支原体的免疫原性基因p36、p46、p65和p97R1-Nrdf克隆到pYA3493,得到重组质粒pYA-36、pYA-46、pYA-65和pYA-97R1-Nrdf。重组质粒和空质粒pYA3493分别电转asd基因缺失株C500ˉ,获得重组菌株C36(pYA-36)、C46(pYA-46)、C65(pYA-65)、C97R1-Nrdf(pYA-97R1-Nrdf)和空质粒菌株CpYA(pYA3493)。研究重组菌株的生物学特性,并以小鼠为动物模型评价重组菌株在口服、肌注两种不同免疫途径下的免疫原性。【结果】成功构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,重组菌株能表达外源蛋白,生化和生长特性未发生改变,插入的外源基因亦稳定存在。小鼠的免疫原性结果显示:口服C36+C46+C65+C97R1-Nrdf组的猪肺炎支原体抗体极显著高于口服C36+C46+C65组和肌注商品疫苗组(P<0.01),但与肌注C36+C46+C65组无显著性差异(P>0.05);IFN-γ为肌注C36+C46+C65组显著高于肌注商品疫苗组(P<0.05),而与口服C36+C46+C65或C36+C46+C65+C97R1-Nrdf组差异均不显著(P>0.05);IL-4水平为口服C36+C46+C65组>口服C36+C46+C65+C97R1-Nrdf组>肌注商品疫苗组>肌注C36+C46+C65组,但各组之间差异均不显著(P>0.05)。对照组的猪肺炎支原体抗体、IFN-γ以及IL-4均与试验组差异极显著(P<0.01)。【结论】构建的表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,采用肌注免疫时具有较好的免疫原性,有望发展为猪肺炎支原体的基因工程疫苗。     

共表达猪瘟病毒E2基因和猪白细胞介素2基因的重组腺病毒的构建及其免疫原性研究

为研制猪瘟活载体疫苗,构建共表达猪瘟病毒E2基因和猪白细胞介素2基因的重组腺病毒并研究其免疫原性。应用PCR方法从pMD19-T-E2质粒和pMD19-T-pIL2质粒中分别扩增E2基因和pIL-2基因,将扩增的全长E2基因和pIL-2的编码区基因序列通过柔性接头序列(5个甘氨酸密码子)串联,插入腺病毒穿梭载体AdTrack中,在受体菌中与骨架载体AdEasy同源重组。重组质粒AdEasy-E2-pIL-2转染HEK293细胞,包装出重组腺病毒rAd-E2-pIL-2。用rAd-E2-pIL-2免疫家兔,经兔体交互免疫试验评定其免疫效果。结果显示,经RT-PCR和West-ern blot检测,成功构建了rAd-E2-pIL-2,重组病毒滴度达108.12PFU/mL。rAd-E2-pIL-2接种家兔后刺激接种兔产生猪瘟病毒特异性抗体,淋巴细胞转化试验结果显示猪瘟病毒诱导兔淋巴细胞特异性增殖,攻毒后rAd-E2-pIL-2接种兔和猪瘟病毒C株接种兔均未出现定型热反应。研究结果表明,rAd-E2-pIL-2免疫兔可以预防猪瘟病毒C株接种引发的体温反应,rAd-E2-pIL-2可望成为猪瘟候选疫苗。     

Suppressive effect of IL-4 on IL-13-induced genes in mouse lung

Although IL-4 signals through two receptors, IL-4R alpha/common gamma-chain (gamma(c)) and IL-4R alpha/IL-13R alpha1, and only the latter is also activated by IL-13, IL-13 contributes more than IL-4 to goblet cell hyperplasia and airway hyperresponsiveness in murine asthma. To determine whether unique gene induction by IL-13 might contribute to its greater proasthmatic effects, mice were inoculated intratracheally with IL-4 or IL-13, and pulmonary gene induction was compared by gene microarray and real-time PCR. Only the collagen alpha2 type VI (Ca2T6) gene and three small proline-rich protein (SPRR) genes were reproducibly induced > 4-fold more by IL-13 than by IL-4. Preferential IL-13 gene induction was not attributable to B cells, T cells, or differences in cytokine potency. IL-4 signaling through IL-4R alpha/gamma(c) suppresses Ca2T6 and SPRR gene expression in normal mice and induces these genes in RAG2/gamma(c)-deficient mice. Although IL-4, but not IL-13, induces IL-12 and IFN-gamma, which suppress many effects of IL-4, IL-12 suppresses only the Ca2T6 gene, and IL-4-induced IFN-gamma production does not suppress the Ca2T6 or SPRR genes. Thus, IL-4 induces genes in addition to IL-12 that suppress STAT6-mediated SPRR gene induction. These results provide a potential explanation for the dominant role of IL-13 in induction of goblet cell hyperplasia and airway hyperresponsiveness in asthma.     

Differential responses of hematopoietic and non-hematopoietic cells to anti-inflammatory cytokines: IL-4, IL-13 and IL-10.

Recombinant preparations of human anti-inflammatory cytokines: IL-4, IL-13 and IL-10, inhibited LPS-induced synthesis of TNFalpha and IL-6 in the whole human blood tested in vitro. These cytokines also inhibited LPS-induced IL-6 and TNF mRNA accumulation in isolated human blood monocytes/macrophages. On the other hand, similar concentrations of IL-4 and IL-13 (but not IL-10) enhanced synthesis of IL-6 in cultured human umbilical vein endothelial cells (HUVEC). In human hepatoma HepG2 cells IL-4 and IL-13 (but not IL-10) inhibited IL-6-induced synthesis of haptoglobin. These differential responses to the tested anti-inflammatory cytokines were observed at mRNA and protein levels and may reflect cell specificities in signalling pathways and gene expression. When HUVEC and HepG2 cells were cultured together and stimulated with LPS the addition of IL-4 or IL-13 resulted in the reduction of LPS-induced and IL-6-mediated haptoglobin synthesis. Thus in co-culture the inhibitory effects of IL-4 or IL-13 on HepG2 cells prevail over stimulation of IL-6 synthesis in HUVEC.     

IL-6 and IL-1 synergize to stimulate IL-2 production and proliferation of peripheral T cells

Purified T cells can be induced to proliferate and to produce the autocrine growth factor IL-2 with mAb to the TCR and costimulatory cytokines. In a previous report we demonstrated that human IL-6 stimulates IL-2 production and proliferation of purified T cells, in conjunction with the insolubilized anti-TCR V beta 8 mAb, F23.1. Here we show that when CD4+ T cells are rigorously purified to greater than 99% CD4+CD8-, they respond only weakly to F23.1 and IL-6. Instead, there is an additional requirement for IL-1, which dramatically synergizes with IL-6 to induce prolonged (greater than 7 days) proliferative responses and IL-2 production. Similar results were observed when the highly mitogenic anti-CD3 mAb 145-2C11 was substituted for F23.1. The proliferation induced by F23.1, IL-1, and IL-6 was substantially (greater than 80%) inhibited by a mAb to mouse IL-2, and was not inhibited by an anti-IL-4-mAb. In accordance with this finding, medium conditioned by the activated CD4+ cells contained large amounts of IL-2, which increased over a 7-day culture period. These results demonstrate that IL-6 and IL-1 stimulate T cell proliferation by inducing production of the autocrine growth factor IL-2. In addition, the two lymphokines must be present simultaneously for activation to occur. The possible roles of IL-6 and IL-1 in IL-2 gene regulation and in Ag-induced T cell activation are discussed.     

Nitric oxide and peroxynitrite induce gene expression of interleukin receptors increasing IL-21, IL-7, IL-1 and oncostatin M in cardiomyocytes

AimThe objective of this investigation was to determine whether nitric oxide (NO) and peroxynitrite alter the gene expression of interleukin receptors (IL-R) in cardiomyocytes.Main methodsCardiomyocytes, from neonatal mouse heart, in culture were treated with the NO donor 3-morpholinosydnonimine hydrochloride (SIN-1), which releases NO and peroxynitrite. IL-R gene expression was assessed by microarray analysis.Key findingsGene expression data show that the IL-2 receptor family showed a highly significant and 1.7-fold increase in the gamma chain component which is common to all members of the IL-2 family receptors. There was a significant and 2-fold increase in IL-21 R and an increase of 1.8-fold in IL-7 Rα gene expression. In contrast there was a significant 0.7-fold decrease in IL-9 Rα. The IL-1 receptor family showed a significant and 1.4-fold increase in IL-1 R1 compared to control but no change in IL-18 R1 gene expression which was similar to control. The IL-6 family showed a significant increase in oncostatin M receptor gene expression of 1.3-fold but no change in IL-6 R alpha or IL-11 R alpha.SignificanceThese data suggest that NO/peroxynitrite by increasing gene expression of certain IL-Rs may magnify the effects of specific interleukins in conditions of excess interleukin production.     

Prostaglandin E2 augments IL-10 signaling and function

In inflamed joints of rheumatoid arthritis, PGE(2) is highly expressed, and IL-10 and IL-6 are also abundant. PGE(2) is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE(2) on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE(2) significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE(2) suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE(2)-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE(2)-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE(2) selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE(2) may modulate immune responses by alteration of cytokine signaling in THP-1 cells.     

Overexpression and structure--function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine.

A synthetic chimeric IL-2/IL-6 gene was synthesized to engineer a bifunctional lymphokine which was overproduced in Escherichia coli. Following denaturation of the inclusion bodies in 6 M guanidine and refolding and reoxidation in the presence of a redox system, the fusion protein (rIL-2/IL-6) was purified to homogeneity and shown to react with both monospecific anti-IL-2 and anti-IL-6 antisera. A collagen-like spacer was introduced between the two cytokine moieties to generate IL-2 and IL-6 molecules upon collagenase digestion. After cleavage, the two subunits, purified in a single-step procedure, were found to be correctly reoxidized and functionally as active as their native counterparts. Circular dichroism studies of rIL-2/IL-6 revealed that both cytokine subunits refolded independently and exhibited the alpha-helical structures characteristic of the corresponding wild-type lymphokines. The chimera displayed full IL-2 activity in the CTLL-2 cell proliferation assay. It also retained the IL-6 property to enhance IgM synthesis in SKW6.4 cells, induce the proliferation of B-cell hybridomas and stimulate the production of fibrinogen in hepatocytes. Because IL-2 amplifies the cellular immune response and IL-6 up-regulates the humoral response, this bifunctional lymphokine represents a potentially useful therapeutic adduct and may serve as an immunomodulator to enhance the host's response to vaccination.