长白猪IL-4基因克隆及真核表达载体的构建

将长白猪外周血淋巴细胞在刀豆素A(ConA)的刺激下培养24 h后,提取其总RNA,应用RT-PcR技术扩增白细胞介素-4(IL-4)cDNA,克隆到pMD19-T载体并测序.将构建好的载体双酶切并回收目的片段与pcDNA3.1(+)载体连接并转化入Top10中构建真核表达载体,真核表达载体经双酶切及测序结果表明:克隆的IL-4 cDNA全长为411个碱基,其中ORF为402个碱基,编码133个氨基酸,与GenBank公布的猪IL-4比对同源性为100%,从而证实成功构建了长白猪IL-4 cDNA真核表达载体.     

荣昌猪白细胞介素-6基因的克隆与序列分析

根据GenBank收录的猪白细胞介素-6(PIL-6)设计1对特异性引物,经刀豆蛋白素A(ConA)诱导猪淋巴细胞并提取总RNA,用RT-PCR方法扩增出荣昌猪IL-6的cDNA。将扩增基因连接到PMD18-T质粒上,经酶切鉴定和序列测定证明该序列是PIL-6。序列分析结果表明:该基因cDNA全长741 bp,开放阅读框由639个核苷酸组成,推测产生的编码产物由212个氨基酸组成。核酸序列分析比对发现:荣昌猪IL-6与GenBank已发表的IL-6序列的同源性较高,为99.8%~100%,氨基酸的同源性为99.5%~100%。对荣昌猪IL-6基因氨基酸的亲水性和蛋白表面可能性进行分析,表明其与IL-6基因氨基酸序列性质一致。     

猪白细胞介素-15基因的克隆及生物信息学分析

参照GenBank登录的猪白细胞介素-15(IL-15)基因序列自行设计1对特异性引物，运用RT PCR技术从由刀豆蛋白A(ConA)诱导培养的荣昌猪外周血淋巴细胞扩增出猪IL-15基因的完整CDS序列，全长489 bp。同源比对结果表明，荣昌猪IL-15基因与已公布的2个猪种IL-15基因核苷酸同源性均为99.4%,与哺乳动物的同源性较高，与鸡的同源性较低。密码子偏爱性分析显示，荣昌猪IL-15基因在密码子使用上存在一定的偏爱性；分子进化分析结果表明其与人及哺乳动物IL-15基因的进化关系较近，而与禽类鸡的IL-15基因进化距离较远；结构域预测分析显示其与人的IL-15存在很大的相似性，可能在功能上有相似性。     

藏猪白细胞介素-2基因的克隆及序列分析

将藏猪外周血淋巴细胞在伴刀豆球蛋白 A(Con A)的刺激下体外培养 70 h后 ,提取激活淋巴细胞总 RNA,应用RT- PCR技术扩增出藏猪淋巴细胞白细胞介素 - 2 c DNA (TPIL - 2 ) ,克隆到 p MD- T载体上并测序。测序结果显示 ,克隆的TPIL- 2 c DNA全长为 5 0 3个碱基 ,开放阅读框 (ORF)为 4 6 5个碱基 ,编码 15 4个氨基酸 ,分子量为 17.4 kd,等电点为 5 .38;疏水氨基酸 4 0 .3% ,亲水氨基酸 37.0 % ,碱性氨基酸 11% ,酸性氨基酸 11.7%。此 c DNA与报道猪的 IL - 2同源性为 10 0 % ,证实克隆到了藏猪白细胞介素 - 2基因 c DNA。     

荣昌猪自细胞介素-6基因的克隆与序列分析

根据GenBank收录的猪白细胞介素-6（PIL-6）设计1对特异性引物，经刀豆蛋白素A（Co—nA）诱导猪淋巴细胞并提取总RNA，用1it—PCR方法扩增出荣昌猪IL-6的cDNA。将扩增基因连接到PMD18-T质粒上，经酶切鉴定和序列测定证明该序列是PIL-6。序列分析结果表明：该基因cDNA全长741bp，开放阅读框由639个核苷酸组成，推测产生的编码产物由212个氨基酸组成。核酸序列分析比对发现：荣昌猪IL-6与GenBank已发表的IL-6序列的同源性较高，为99．8％。100％，氨基酸的同源性为99．5％-100％。对荣昌猪IL-6基因氨基酸的亲水性和蛋白表面可能性进行分析，表明其与IL-6基因氨基酸序列性质一致。     

长白猪IL-2基因克隆及真核表达载体的构建

为了对长白猪白细胞介素-2(IL-2)基因的生物学作用进行研究,试验将长白猪外周血淋巴细胞在刀豆素A(ConA)的刺激下培养24 h后,提取总RNA,应用RT-PCR技术扩增IL-2 cD-NA,克隆到pMD19-T载体并测序。对构建好的T载体双酶切并回收目的片段,将回收片段与pcD-NA3.1(+)载体连接并转化到Top 10中构建真核表达载体。结果表明:克隆的IL-2 cDNA全长为526 bp,ORF全长为465 bp,编码154个氨基酸,与GenBank公布的猪IL-2比对同源性为100%,进而证明成功地克隆了长白猪IL-2 cDNA;并且真核表达载体双酶切及测序表明成功构建了长白猪IL-2真核表达载体。     

广西沼泽型水牛白细胞介素-10和干扰素-γ基因的克隆及其序列分析

将本地沼泽型成年水牛外周血单核细胞(PBMC)在刀豆素A(ConA)的刺激下体外培养,提取培养的单核细胞总RNA,应用逆转录聚合酶链式反应(RT-PCR)技术扩增水牛白细胞介素-10(IL-10)和干扰素-γ(IFN-γ),并克隆到pMD18-T载体上,用EcoRⅠ和HindⅢ双酶切鉴定筛选阳性克隆,然后进行测序。序列分析结果显示:IL-10开放阅读框(ORF)由536个核苷酸组成,共编码178个氨基酸;在核苷酸水平上与印度水牛、牛、绵羊、猪、马、人和鼠的IL-10基因的同源性为75.9%～99.6%;氨基酸的同源性为74.2%～100%。IFN-γ的ORF由501个核苷酸组成,共编码166个氨基酸;在核苷酸水平上与印度水牛、牛、绵羊、猪、马、人和鼠的IFN-γ基因的同源性为60.7%～99.6%;氨基酸的同源性为40.6%～100%。     

鸡白细胞介素15基因的克隆及序列分析

根据GenBank中所收录的鸡白细胞介素15(ChIL 15)的cDNA序列设计特异性引物,以经ConA刺激3 h的科宝鸡脾脏淋巴细胞的总RNA 为模板,用RT PCR 方法克隆获得了ChIL 15的cDNA。ChIL 15的cD NA全长655 bp,其中第84 位~644 位是该基因的阅读框(ORF),共编码187 个氨基酸。将所克隆的序列与已报道序列相比较,二者核苷酸的同源性为100%(655/655),所编码蛋白质的氨基酸序列同源性也为100%。     

杂交猪白细胞介素-18全基因的克隆与序列分析

通过RT-PCR方法直接从猪脾脏淋巴细胞中扩增出猪白细胞介素-18(IL-18)全基因的cDNA,其大小为579bp,编码192个氨基酸。与GenBank上已发表猪IL-18序列(ABO10003)进行比较,核苷酸同源性为99.8%,在第550位处(以ATG为1计)由A→G,存在有意义突变。与GenBank上的ABO10003、AF176949、AY262109、NM1997序列进行比较分析,氨基酸同源性分别为99%,98.5%,99.8%和99%。从系统进化树可以看出,河南良杂猪IL-18基因与ABO10003、AY262109亲缘关系最近。河南良杂猪IL-18基因和人及其他动物IL-18基因核苷酸序列进行比较分析,结果显示猪与人、猫、牛、鸡、鸭、海豚、山羊、马、家鼠、老鼠、绵羊的IL-18基因核苷酸同源性分别为83.1%、88.3%、90.7%、26.8%、31.4%、26.3%、90.0%、91.5%、67.7%、65.1%和90.8%。     

贵州白香猪白细胞介素4全基因的克隆与序列分析

为获得贵州白香猪白细胞介素4的基因,以提取的经刀豆蛋白A诱导培养的贵州白香猪外周血淋巴细胞总RNA为模板,应用RT-PCR技术扩增全长猪白细胞介素4(Interleukin-4,IL-4)基因,并克隆到pM D18-T Simple Vector载体后测序。测序结果表明:IL-4基因的ORF为402 bp,可编码133个氨基酸,其中前24个为信号肽序列,后109个氨基酸为成熟肽,具有3个潜在的N-糖基化位点;贵州白香猪除与AF493991(99.4%),L12991(98.1%)氨基酸一致性相对较低外,与其他猪源IL-4基因氨基酸一致性均为100%,而与其他种属动物的IL-4基因氨基酸一致性则较低,为37.2%~82.7%。     

A monoclonal antibody to equine interleukin-4

Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1–2% of lymphocytes using flow cytometric analysis. In the presence of the secretion blocker Brefeldin A, the protein accumulated and was detected in 4–8% of lymphocytes stimulated with phorbol 12-myristate 13-acetate and ionomycin. Double staining with the new mAb and T-cell or B-cell markers identified a subpopulation of CD4⁺ T-cells expressing the protein recognized by the mAb. In addition, the protein was detectable in cell culture supernatants of mitogen stimulated cells by ELISA when using the new mAb for coating of the plates and a polyclonal antiserum to equine IL-4 for detection. In conclusion, the new mAb detects equine IL-4 and can be used for intracellular staining and ELISA to measure this important cytokine.     

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16 kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.     

Cloning and expression of recombinant equine interleukin-3 and its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes

Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL).Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culicoides nubeculosus extract with or without IL-3. Sulfidoleukotriene (sLT) production was measured in supernatants by ELISA and mRNA expression of IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) assessed in cell lysate by quantitative real-time PCR.Recombinant equine IL-3 (req-IL-3) had a dose dependent effect on sLT production by stimulated equine PBL and significantly increased IL-4, IL-13 and TSLP expression compared to non-primed cells.IL-3 priming significantly increased Culicoides-induced sLT production in IBH-affected but not in non-affected horses and was particularly effective in young IBH-affected horses (≤3 years).A functionally active recombinant equine IL-3 has been produced which will be useful for future immunological studies in horses. It will also allow improving the sensitivity of cellular in vitro tests for allergy diagnosis in horses.     

猪IL-15 cDNA真核表达载体的构建、表达及其免疫佐剂效应

参考GenBank发表猪IL-15mRNA序列设计引物,用RT-PCR方法扩增猪IL-15cDNA,并克隆到pMD18-T载体中,通过PCR、酶切和测序验证克隆正确,再亚克隆到真核表达载体pcDNA-3.1（＋）上,得到重组质粒pcD-NA-pIL-15。在脂质体介导下,重组质粒pcDNA-pIL-15转染AD-293细胞。以鼠抗猪IL-15为一抗,用间接免疫荧光分析表明猪IL-15基因均在AD-293细胞中成功进行了瞬时表达。小鼠免疫试验表明,pcDNA-pIL-15作为免疫佐剂,能够有效提高小鼠的脾T细胞增殖,加强pcDNA-ORF2（PCV2）质粒免疫过程中的特异性中和抗体的产生,为进一步研制猪IL-15基因佐剂疫苗及进行临床实验奠定基础。     

Effect of transgenic expression of porcine interleukin-6 gene and CpG sequences on immune responses of newborn piglets inoculated with Pseudorabies attenuated vaccine

Porcine interleukin-6 gene and CpG sequences were used as immunoadjuvants to enhance the immune responses of newborn piglets to Pseudorabies attenuated vaccine (PAV). The titer of specific antibodies to PAV, the proliferation of lymphocytes and induced IL-2 activities were all examined to identify the immune response of the piglets. The results showed that the immune responses with CpG ODN and porcine interleukin-6 gene were significantly stronger than routine immunities. The data suggests that porcine IL-6 and CpG motifs could be employed as effective immunoadjuvants to raise the humoral and cellular responses of newborn piglets to Pseudorabies attenuated vaccine.     

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.     

Relationships among peripheral eosinophilia, eosinophil peroxidase activity, interleukin-5 concentration and faecal nematode egg count during natural, mixed gastrointestinal nematode infection

A longitudinal study of sheep, naturally infected with gastrointestinal helminths, was carried out to quantify the relationships among eosinophilia, eosinophil peroxidase activity, interleukin-5 level and faecal egg counts. Faecal egg counts, peripheral eosinophilia and peroxidase activity per eosinophil were moderately repeatable but interleukin-5 concentrations had a remarkably high repeatability. Animals with higher concentrations of interleukin-5 had greater peripheral eosinophilia and those animals with higher numbers of eosinophils had lower faecal egg counts. These associations were statistically significant but quite weak. Variation in interleukin-5 levels does not appear to be responsible for most of the variation in eosinophil responses in outbred sheep.     

Clinical improvement in feline herpesvirus 1 infected cats by oral low dose of interleukin-12 plus interferon-gamma

Feline herpesvirus 1 (FHV-1) is a widespread cat pathogen inducing rhinitis, conjunctivitis and corneal ulcers. To alleviate acute FHV-1-induced disease, antiviral agents are used often with antibiotics. But sometimes, these treatments, as well as conventional doses of cytokines have moderate efficacy and/or collateral effects. Herein we have investigated the effects of low dose interleukin (IL)-12 plus interferon (IFN)-gamma, prepared by Sequential Kinetic Activated (SKA), on the treatment of FHV-1 infection. Twenty-five, unvaccinated FHV-1-positive cats were recruited into a prospective, randomized, placebo-controlled, double-blinded clinical trial. Fifteen cats were treated for 6 months with oral low doses of SKA IL-12 plus IFN-gamma and 10 cats were treated with placebo. At 1, 6 and 12 months (follow-up) after the beginning of treatment, clinical assessment, PCR assay and blood count were carried out. At follow-up, in treated group, we observed significant (p < 0.05) improvements in clinical signs and PCR became negative in 12/15 cats (80%). In placebo, 10/10 cats were PCR-positive, with improvements (30%) or worsening (70%) in clinical signs. Blood values were normal in both groups.Our results show that the low dose therapy, based on activated solutions of IL-12 plus IFN-gamma, represents a novel approach to treat FHV-1 infection in cats.