人白介素12亚基p40和p35基因串联在毕赤酵母中的表达及表达产物活性分析

摘 要 利用毕赤酵母系统表达有活性的人单链白细胞介素12（hscIL-12）,PCR法从质粒pBI121-IL-12中扩增hscIL-12基因,经酶切、连接构建重组表达载体pPIC9K-hscIL-12,SacI线性化后,PEG1000法转化毕赤酵母GS115,经G418筛选和菌落PCR鉴定,经甲醇诱导,hscIL-12在酵母中获得分泌表达,表达产物经Western Blot检测,显示该蛋白相对分子质量为70KDa,可与鼠抗人IL-12单克隆抗体特异性结合;定量分析结果表明,重组酵母培养上清中hscIL-12约占总蛋白的26％,表达量约为60mg／L;生物学活性实验表明,重组蛋白能促进人外周血淋巴细胞增殖。为利用rhscIL-12进行基因治疗奠定了基础。     

猪IL-18 在杆状病毒/昆虫细胞中的表达

IL-18作为一种多效性细胞因子,在机体免疫应答及各种生理功能中发挥着重要的调节作用,根据猪IL-18基因序列设计1对引物,将编码猪IL-18的成熟蛋白基因亚克隆到杆状病毒转移载体pFastBacDual中,并在C端融合6个组氨酸标签以利于纯化,然后转化到含穿梭载体Bacmid的感受态细胞DH10Bac中,发生转座作用。将重组质粒转染昆虫细胞,SDS-PAGE可检测到分子量为18 kDa左右的重组蛋白,Western blotting证实该重组蛋白可与兔抗猪IL-18抗体发生特异性反应。纯化后蛋白能明显促进猪T淋巴细胞转化,表明所表达的IL-18具有较高的生物活性。此研究为进一步开发研制新型免疫佐剂奠定了基础。     

重组线虫抗凝肽在毕赤酵母中的表达

目的：获得具有抗凝活性的重组线虫抗凝肽（NAP）蛋白, 为新型抗凝药物的开发奠定基础。方法：将pPIC3.5K-rNAP质粒转化毕赤酵母菌株GS115,筛选出阳性菌株后,用甲醇诱导表达。收集酵母培养上清,用SDS-PAGE和Western blot分析表达产物,通过测定PT(血浆凝血酶原时间)、INR（血浆凝血酶原国际标准化比率）和APTT（活化部分凝血活酶时间）来验证其生物学活性。结果：获得了稳定表达NAP的重组酵母菌株。重组NAP被分泌表达,其分子量比预计分子量（8.7KD）稍大,约为10KD左右,可能与糖基化有关。体外活性检测证实其有较强的抗凝活性。结论：在毕赤酵母中成功表达了具有生物学活性的重组线虫抗凝肽,可用于新型抗凝药物的开发。     

猪IL-18在乳酸乳球菌中的表达及其生物活性的检测

为了在乳酸乳球菌中分泌表达具有生物活性的猪IL-18蛋白,并检测其生物活性,故通过分离猪外周血单核淋巴细胞(PBMC),以其为模板,采用RT-PCR方法扩增猪白细胞介素18(pIL-18)基因,将目的基因与乳酸乳球菌表达载体pAMJ399进行连接,并电转化至乳酸乳球菌MG1363中,通过SDS-PAGE和Western blotting分析检测目的蛋白的表达,并通过脾淋巴细胞增殖试验和细胞病变抑制法对pIL-18的生物活性进行检测。Western blotting分析检测结果与生物活性检测结果显示,在重组菌pAMJ399-pIL18/MG1363的上清和菌体沉淀中19 kDa处均出现pIL-18的特异蛋白反应带,且分泌表达的pIL-18蛋白能明显促进猪脾淋巴细胞的增殖,并对病毒增殖有明显的抑制作用。以上结果表明pIL-18可在乳酸乳球菌分泌表达,且表达产物具有良好的生物活性。     

白细胞介素-21与乙型肝炎病毒前S2S抗原融合蛋白在293T细胞中的表达

目的:构建白细胞介素-21(interleukin-21,IL-21)和乙型肝炎病毒前S2S抗原(S2S)的融合表达质粒,并研究其在293T细胞中的表达。方法:采用PCR方法扩增IL-21和HBV前S2S基因片段,分别克隆入pcDNA3真核表达质粒,用分子克隆方法构建融合表达质粒,并以脂质体2000转染293T细胞,分别应用ELISA法和Western Blot法检测细胞上清及细胞中IL-21和HBsAg的表达水平。结果:经酶切鉴定及DNA序列证实重组质粒内插入片段序列正确,三种重组质粒分别命名为pcDNA-IL-21、pcDNA-S2S和pcDNA-IL-21-S2S,并且重组质粒能在293T细胞内表达并分泌相关蛋白。结论:成功构建IL-21和乙型肝炎病毒前S2S抗原的融合表达质粒,重组质粒能在真核细胞内表达。     

Expression of equine herpesvirus 1 glycoprotein D by using a recombinant baculovirus.

Glycoprotein D (gD) of equine herpesvirus 1 (EHV-1) was expressed at the surface of insect cells infected by a recombinant baculovirus. EHV-1 gD was detected as multiple forms (56, 52, and 48 kDa) from 18 to 96 h postinfection. Laboratory animals inoculated with the recombinant EHV-1 gD developed neutralizing antibody responses against both EHV-1 and EHV-4.     

共表达HIV-1中国流行株gp120与IL-18重组鸡痘病毒的构建及其免疫原性观察

为筛选我国的HIV-1疫苗候选株,以鸡痘病毒282E4中国疫苗株为载体,构建了共表达中国流行株HIV-1外膜蛋白gp120和IL-18的重组鸡痘病毒,并将该重组鸡痘病毒免疫BALB/c小鼠,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体水平。结果显示,HIV_1外膜蛋白gp120和IL-18不但可在重组鸡痘病毒感染的鸡胚成纤维细胞中表达,而且可在重组鸡痘病毒感染的哺乳动物细胞中表达。重组病毒具有良好的免疫原性,可诱导小鼠产生特异性抗体和脾特异性CTL反应,且IL_18发挥了免疫佐剂的作用。本研究结果为制备安全、有效的HIV-1基因工程活载体疫苗奠定了基础。     

Studies on the structure and regulation of the human hepatic interleukin-6 receptor

Affinity cross-linking of 125I-labeled recombinant human interleukin-6 (IL-6) to human hepatoma cells (HepG2) allowed the detection of three IL-6-containing complexes with molecular masses of 100 kDa, 120 kDa and 200 kDa. Treatment of HepG2 cells with dexamethasone led to a time- and dose-dependent up-regulation of IL-6-receptor mRNA levels. By the use of cross-linking this effect was also seen at the protein level, where all three IL-6-binding complexes increased upon incubation of HepG2 cells with dexamethasone. Under conditions of IL-6-receptor up-regulation by dexamethasone, gamma-fibrinogen mRNA induction by IL-6 is stronger and occurs earlier than without dexamethasone. We propose therefore that the expression of the IL-6 receptor might be a rate-limiting step in acute-phase-protein induction.     

Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.     

猪繁殖与呼吸综合征病毒nsp4抗体制备与鉴定

为了获得猪繁殖与呼吸综合征病毒(PRRSV)nsp4的抗体,根据HP-PRRSV TA-12株(Gen Bank Accession No.HQ416720)的nsp4基因序列,设计并合成一对引物。用RT-PCR扩增后克隆到原核表达载体p ET-28a(+)中,构建重组质粒p ET28a-nsp4,转化至Trasseta(DE3),经IPTG诱导重组蛋白获得了高效可溶性表达,大小约为26 k Da。经镍离子亲和柱(Ni+-NTA)纯化获得了高纯度重组蛋白,将纯化的nsp4蛋白免疫新西兰大白兔制备了多克隆抗体。ELISA检测抗体效价可达106,Western blotting和IFA检测结果表明所制备的多克隆抗体具有良好的免疫反应特异性,能够识别PRRSV感染宿主细胞中的nsp4蛋白。本研究成功制备了针对nsp4的多克隆抗体,为进一步研究nsp4的功能及PRRSV致病机制奠定了基础。     

Cloning, sequencing, and expression of porcine interleukin-18 in Escherichia coli

IL-18 is the new name of a novel cytokine that plays an important role in T(H1) response, primarily by its ability to induce IFN-gamma production in T cells and natural killer cells. The porcine IL-18 gene was isolated using RT-PCR from porcine alveolar macrophages. Sequence analysis of the porcine IL-18 gene has demonstrated an open reading frame of 579 base pairs encoding 192 amino acids precursor protein with a predicted molecular mass of 22 kDa. The porcine IL-18 gene shares 84% and 89% similarity to the human and canine equivalents, respectively, at the nucleotide level. The cloned IL-18 was expressed in Escherichia coli and its expression was confirmed by SDS-PAGE and Western blotting.     

Nitric Oxide Inducing Function and Intracellular Movement of Chicken Interleukin-18 in Cultured Cells

To evaluate the characteristics of chicken interleukin-18 (ChIL-18) in different forms in vitro,the ChIL-18 full-length gene (ChIL-18-F) and the ChIL-18 presumed mature protein gene (ChIL-18-M) werecloned and inserted into the eukaryotic expression vector pCI,to construct recombinant pCI-ChIL-18-Fand pCI-ChIL-18-M.The recombinant plasmids were then transferred into chicken splenic lymphocytes(CSLs).Western blot showed that ChIL-18-F,with a molecular weight of 23.0 kDa,was produced in CSLstransfected by pCI-ChlL-18-F;ChIL-18-M,with a molecular weight of 19.5 kDa,was produced in CSLstransfected by pCI-ChIL-18-M.The nitric oxide (NO) level in the transfected CSLs and the culture mediumat different time points was further examined under confocal microscopy using 4.5-diaminofluoresceinstaining.The results showed that both pCI-ChIL-18-F and pCI-ChIL-18-M groups showed significantincrease in intracellular and extracellular NO production compared with pCI transfected control cells.Theseresults suggest that both ChIL-18-F and ChIL-18-M could stimulate NO secretion in CSLs.To characterizethe intracellular distribution of ChIL-18,ChIL-18-F and ChIL-18-M were each fused to the enhanced greenfluorescent protein gene,and expressed in Vero cells.The results showed that the ChIL-18-F tended to themembranous region in Vero cells,while ChIL-18-M did not.This indicates that the N-terminal 27 amino acidpeptide helped ChIL-18 target to Veto cell membranes.     

Nitric oxide inducing function and intracellular movement of chicken interleukin-18 in cultured cells

To evaluate the characteristics of chicken interleukin-18 （ChIL-18） in different forms in vitro, the ChIL-18 full-length gene （ChIL-18-F） and the ChIL-18 presumed mature protein gene （ChIL-18-M） were cloned and inserted into the eukaryotic expression vector pCI, to construct recombinant pCI-ChIL-18-F and pCI-ChIL-18-M. The recombinant plasmids were then transferred into chicken splenic lymphocytes （CSLs）. Western blot showed that ChIL-18-F, with a molecular weight of 23.0 kDa, was produced in CSLs transfected by pCI-ChIL-18-F; ChIL-18-M, with a molecular weight of 19.5 kDa, was produced in CSLs transfected by pCI-ChIL-18-M. The nitric oxide （NO） level in the transfected CSLs and the culture medium at different time points was further examined under confocal microscopy using 4,5-diaminofluorescein staining. The results showed that both pCI-ChIL-18-F and pCI-ChIL-18-M groups showed significant increase in intracellular and extracellular NO production compared with pCI transfected control cells. These results suggest that both ChIL- 18-F and ChIL- 18-M could stimulate NO secretion in CSLs. To characterize the intracellular distribution of ChIL-18, ChIL-18-F and ChIL-18-M were each fused to the enhanced green fluorescent protein gene, and expressed in Vero cells. The results showed that the ChIL-18-F tended to the membranous region in Veto cells, while ChIL- 18-M did not. This indicates that the N-terminal 27 amino acid peptide helped ChIL-18 target to Vero cell membranes.     

鸭白介素-18基因重组真核表达载体的构建及其表达产物的生物学活性

从克隆质柱pGEM-DuIL-18扩增出鸭IL-18全基因片段,克隆到真核表达载体pcDNA3.1( )中,构建重组质粒pcDNA3.1/DuIL-18(简称pDuIL-18).将重组质粒pDuIL-18转染Cos7细胞,转染细胞中含鸭IL-18基因的mRNA.SDS-PAGE分析表明,表达产物是与鸭IL-18相符的约23 000的蛋白条带.鸭淋巴细胞转化试验表明,表达产物对鸭淋巴细胞具有明显诱导转化作用.重组质柱pDuIL-18对H<,9>亚型禽流感灭活疫苗免疫增强作用的研究表明重组质粒pDuIL-18能够提高禽流感灭活疫苗诱发的细胞免疫应答,为研究能够更好地防制禽流感的新型疫苗提供了新的思路.     

In vitro analyses of tissue structure and interleukin-1beta expression and production by human oral mucosa in response to Candida albicans infections

Clinical and experimental observations suggest that oral epithelial cells play a key role in host defenses against candidal infections through cytokines and chemokines. We thus attempted to determine whether oral epithelial cells convey IL-1beta as a pro-inflammatory cytokine in response to Candida albicans infections. We created engineered human oral mucosa (EHOM), put them in contact with live and heat-inactivated C. albicans (10(5) yeast/cm2), and measured the expression of IL-1beta mRNA and protein. Tissue structure and C. albicans morphology were also evaluated. Only live C. albicans modulated IL-1beta expression and secretion. IL-1beta mRNA expression significantly increased during the early stages of infection and decreased during the later stages. The modulatory effect of C. albicans on IL-1beta expression was confirmed by the fact that increased amounts of inactive IL-1beta (33 kDa) were detected early during the infection which then dropped dramatically. There was a significant and time-dependent increase in the amount of the active form of IL-1beta (17 kDa) secreted into the supernatant by epithelial cells infected with live C. albicans. Histological features revealed damage to infected tissues (separation of epithelial cells, edema, vacuolization, reduction in thickness) compared to uninfected ones. Morphological analyses showed that C. albicans changed from a blastospore to a hyphal form at later infection periods. This transformation was very pronounced at 8 and 24 h post-infection. These results provide additional evidence for the contribution of oral epithelial cells to local defenses against exogenous stimulations such as C. albicans infections.     

KLF4siRNA慢病毒载体构建和鉴定

目的:构建上皮锌指蛋白4(Krüppel-like factor 4,KLF4)siRNA慢病毒载体并进行初步鉴定,为研究KLF4在宫颈细胞癌中的分子机制奠定基础。方法:利用公用网站中提供的RNA干扰序列设计原则,设计4个RNA干扰靶点序列,合成含干扰序列的单链DNA oligo,然后退火配对产生双链,再通过其两端所含酶切位点直接连入酶切后的RNAi慢病毒载体上;将连接产物转入制备好的细菌感受态细胞,PCR鉴定阳性重组子后,送测序验证,测序结果经比对确认正确的克隆,制备编码慢病毒颗粒的重组病毒质粒及其两种辅助包装原件载体质粒,共转染293T细胞,收集富含慢病毒颗粒上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。收集上清液感染宫颈癌He La细胞,通过q RT-PCR及Western Blot鉴定KLF4 siRNA慢病毒干扰效果。结果:成功构建KLF4 siRNA慢病毒载体。KLF4 siRNA慢病毒感染He La细胞后,q RT-PCR及Western Blot测定结果显示,KLF4表达明显降低。结论:KLF4 siRNA慢病毒载体构建及包装成功,可有效抑制KLF4表达,为研究KLF4生物学功能奠定基础。     

人视黄醇结合蛋白4在杆状病毒系统中的表达及其多克隆抗体制备

旨在利用杆状病毒系统表达、制备人视黄醇结合蛋白(RBP4)并检测其免疫原性。将人RBP4基因片段及信号肽SS64片段亚克隆到杆状病毒转移载体pFastBac-dual(pFBd)中,获得相应的重组转移质粒;转化大肠杆菌菌株DH10bac,转座后经筛选获得重组穿梭质粒rbacmid,将重组穿梭质粒转染孔板培养的Sf9细胞,获得含人RBP4表达框的重组杆状病毒,经过扩增获得毒种。毒种感染对数生长期的Sf9细胞并表达人RBP4蛋白(I-RBP4),通过SDS-PAGE和Western blotting对表达蛋白进行检测和鉴定。用毒种感染悬浮培养的Sf9细胞制备一批RBP4蛋白,完成SDS、Western blotting的检测及少量的多抗制备。纯化重组蛋白并与E.coli重组人RBP4(E-RBP4)分别免疫家兔。实验结果,酶切鉴定及测序证实重组转移质粒构建正确;成功构建重组RBP4-bacmid;人RBP4蛋白在昆虫细胞获得高效表达。表达的RBP4蛋白可以分泌到培养基中,分子量约为23 kDa,经过计算表达量为100 mg/L;纯化蛋白免疫兔子制备了多抗血清,血清滴度为1∶100 000,高于原核表达的抗体滴度(1∶10 000),与人体提纯蛋白制备的抗体滴度相近。杆状病毒系统高效表达了人的RBP4蛋白,具有较好的抗原性,并获得高亲和力的抗血清,为下一步的人血RBP4检测试剂盒的制备打下了坚实的基础。     

Expression, purification, and characterization of secreted recombinant human insulin-like growth factor-binding protein-6 in methylotrophic yeast Pichia pastoris

The mitogenic and metabolic activities of insulin-like growth factors (IGF) are modulated by a family of six high-affinity IGF-binding proteins (IGFBPs). This study describes the secretion and purification of the recombinant human IGFBP-6 expressed in methylotrophic yeast Pichia pastoris. In this research, a multicopy expression plasmid pA-O815/3xIGFBP-6 containing 3 copies of human IGFBP-6 expression cassette was constructed and transformed into P. pastoris GS115. The encoding sequence of alpha-factor leading peptide fused in-frame at the 5' end of human IGFBP-6 open reading frame and led expressed IGFBP-6 into the secretory pathway. After transformed cells were induced with methanol, medium supernatant was analyzed by SDS-PAGE and Western blotting. The two major protein bands of approximately 30 and approximately 18kDa were detected. The protein of approximately 30kDa was confirmed to be the glycosylated recombinant human IGFBP-6 (rhIGFBP-6), which was partially proteolyzed by protease Kex2 to produce a approximately 18kDa fragment. Approximately 95% homogeneity of the soluble form of 30kDa rhIGFBP-6 were achieved by two-step purification procedure using ion-exchange chromatography and then hydrophobic-interaction chromatography. The rhIGFBP-6 could be distributed to all of the cell body when cultured MDA-MB-231 cell with rhIGFBP-6 and the activities of rhIGFBP-6 were assayed by [(3)H]thymidine incorporation, which revealed that rhIGFBP-6 inhibited IGF-II-stimulated cell proliferation. Our results demonstrated that functional rhIGFBP-6 can be produced in sufficient quantities by using P. pastoris for further structural and functional studies.     

Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line.     

Heterogeneity among human nasopharyngeal carcinoma cell lines for inflammatory cytokines mRNA expression levels.

Using polymerase chain reaction (PCR), we confirmed the expression of interleukin-1 alpha (IL-1 alpha) by the human nasopharyngeal carcinoma (NPC) cell line C15 without contribution of either human IL-1 beta or mouse IL-1 alpha in the biological activity previously found in C15. However we showed that IL-1 alpha was not expressed in all NPCs. IL-1 beta and/or tumor necrosis factor (TNF)-alpha genes could also be activated, independently from the number of Epstein Barr Virus (EBV) copies harbored by the cells. Interestingly, the primary tumor C15 showed a profile of TNF-sensitive tumor while C17, C18 and C19 which were derived from metastasis have a typical profile of TNF-resistant cells. Furthermore, the inflammatory cytokines whose genes are classically induced by IL-1 and TNF were found expressed only in C17 and C19 suggesting another level of heterogeneity among NPCs.