重组人白细胞介素-1α在毕赤酵母中的表达、纯化及生物学活性检测

在毕赤酵母中分泌表达重组人白细胞介素-1α(rh IL-1α),优化rh IL-1α的发酵工艺及纯化方法,以获得高表达、高纯度具有生物学活性的rh IL-1α。通过PCR扩增获得h IL-1α基因,构建其真核表达载体p PICZαA/h IL-1α,电转化至毕赤酵母X-33,用PCR和SDS-PAGE方法筛选高效表达rh IL-1α的工程菌株并进行Western blot鉴定,DEAE弱阴离子离子交换层析一步纯化表达产物,并用MTT法初步检测其对人肝癌细胞7402的生物学作用。rh IL-1α在摇瓶规模下,经甲醇诱导4 d后表达量约为30 mg/L。Western blot检测rh IL-1α的特异性结合,获得纯度约95%,收率40%左右的rh IL-1α,并证明rh IL-1α能够抑制人肝癌细胞7402的增殖。构建了重组h IL-1α的基因工程菌,并在毕赤酵母中实现了高效表达,为进一步研究其生物学活性和功能奠定了基础。     

人表皮生长因子的原核优势表达与复性

目的:在原核系统中获得具有活性和高表达量的重组人表皮生长因子(rh EGF)。方法:对h EGF编码全序列进行优化,构建原核表达载体p ET-24b-h EGF,在大肠杆菌中表达rh EGF;对rh EGF包涵体进行复性,获得具有较高生物活性的重组蛋白。结果:构建了携带159 bp h EGF基因的表达载体p ET-24b-h EGF,rh EGF在原核系统内得到高表达,重组蛋白相对分子质量为6×103,表达量可占细菌总蛋白的15%,包涵体复性率达90%。结论:对h EGF的编码序列进行了优化,实现了其在原核系统内的高表达,通过复性获得了具有良好生物活性的rh EGF。     

草鱼IL-10单克隆抗体的制备与鉴定

白细胞介素10(Interleukin-10, IL-10)参与机体免疫应答调节, 协同其他细胞因子维持免疫系统稳态。为探究草鱼(Ctenopharyngodon idella)IL-10的细胞来源, 研究利用大肠杆菌(Escherichia coli, E. coli)表达系统制备了高纯度的草鱼IL-10重组蛋白, 免疫小鼠制备单克隆抗体后通过免疫印迹、激光共聚焦和流式细胞术对抗体进行分析。结果表明, 获得的单克隆抗体不仅能特异识别大肠杆菌表达的CiIL-10重组蛋白, 而且能识别HEK293细胞中表达的真核IL-10重组蛋白。脂多糖(Lipopolysaccharide, LPS)和草鱼白细胞介素1β(IL-1β)刺激草鱼性腺细胞系(GCO)36h的免疫荧光染色分析表明, 草鱼IL-10单克隆抗体能与草鱼内源IL-10结合, 但IL-10阳性细胞数量在LPS处理前后无明显变化。该研究成功制备了高纯度CiIL-10重组蛋白, 获得了特异性好的高质量单克隆抗体, 为研究草鱼ILC2和Th2细胞的增殖、分化和功能奠定了基础。     

鼻咽癌CD44抑制表达细胞株的建立

目的:构建CD44新剪接变异体siRNA质粒表达载体,建立CD44新变异体抑制表达的鼻咽癌细胞株.方法:合成CD44新变异体特异性干扰DNA片段,干扰DNA片段亚克隆于带绿色荧光蛋白的pGenesil -1.3质粒表达载体中,双酶切和测序鉴定重组表达质粒载体;采用脂质体将重组表达质粒载体转染入鼻咽癌5 -8F细胞系,进行G418筛选;Western blot分析CD44表达.结果:重组CD44干扰DNA片段质粒表达载体的碱基序列和插入方向正确;细胞转染效率达70％;G418筛选获得GFP表达的单克隆鼻咽癌5 -8F细胞株;Western blot分析表明CD44表达受抑制.结论:建立了CD44新变异体抑制表达的鼻咽癌细胞株,为CD44新变异体在鼻咽癌中的生物学功能提供了基础.     

阳离子交换高效液相色谱检测重组人源化抗CD52单克隆抗体的电荷异质性

目的建立阳离子交换高效液相色谱检测重组人源化抗CD52单克隆抗体(monoclonal antibody,m Ab)电荷异质性的方法,并对其进行验证。方法基于m Ab等电点呈弱碱性,且因为抗体所带电荷的不同导致其与色谱柱结合力的不同,采用"PropacTMWCX-10"弱阳离子交换柱,利用盐梯度洗脱的方法依次洗脱出m Ab的酸性电荷变异体、中性电荷变异体及碱性电荷变异体;对方法的专属性、线性、准确性、精密度和耐用性进行验证。结果阳离子交换高效液相色谱分析重组人源化抗CD52 m Ab各电荷变异体的保留时间在14~21 min之间,其中酸性电荷变异体的保留时间在14~17 min,中性电荷变异体的保留时间在17~18 min,碱性电荷变异体的保留时间在18~21 min之间。专属性验证显示空白辅料对照色谱图在样品出峰位置无干扰。线性验证显示酸性电荷变异体、中性电荷变异体和碱性电荷变异体相关系数R2均0.99。准确性验证显示酸性电荷变异体、中性电荷变异体和碱性电荷变异体回收率分别为99.3%~99.9%、99.8%~100.4%和99.3%~99.7%。仪器重复性、样品重复性和中间精密度验证中,各电荷变异体的RSD均5%。耐用性验证中,流动相p H在8.0±0.1、流动相中盐浓度为100 mmol/L±5 mmol/L、柱温在(30±2)℃变化时各电荷变异体的RSD均5%。结论阳离子交换高效液相色谱可以有效分离重组人源化抗CD52 m Ab的酸性电荷变异体、中性电荷变异体和碱性电荷变异体,且该方法具有良好的专属性、线性、准确性、精密度和耐用性。     

人类白细胞介素-2与白细胞介素-7对体外诱导抗原肽SLYNTVAT特异性T淋巴细胞的影响

目的:优化人类白细胞介素-2(rh IL-2)与白细胞介素-7(rh IL-7)在体外诱导培养SLYNTVAT(SL9)抗原特异性T细胞时的用量。方法:分离健康人外周血PBMC作为效应细胞,培养T2(TAP缺陷T淋巴细胞)细胞并加载HIV来源抗原肽作为刺激细胞。SL9抗原肽四聚体检测不同细胞因子用量下特异性T细胞诱导频率大小。结论:rh IL-7在诱导SL9抗原特异性T细胞中起到关键促进作用,SL9特异性T淋巴细胞能够自身分泌大量rh IL-2,减少rh IL-2用量可为体外诱导培养SL9特异性T淋巴细胞提高效率。     

GST-IL-21融合蛋白在大肠杆菌中的表达及其活性测定

目的:克隆人白细胞介素21(IL-21)编码区的cDNA,在大肠杆菌中得以表达,并检测其促进人外周血单核细胞(PBMC)增殖的生物学活性。方法:利用基因工程技术,以植物血凝素(PHA)刺激的人扁桃体细胞cDNA文库为模板,经PCR扩增获得IL-21的编码基因,并将其重组于表达载体pGEX4T-2中,转化大肠杆菌DH5α进行诱导表达,纯化得到GST-IL-21重组融合蛋白;MTT法检测其对促进PBMC增殖的功能。结果:获得了IL-21编码区的cDNA克隆;SDS-PAGE显示经IPTG诱导表达的该融合蛋白相对分子质量为41000;纯化后的GST-IL-21融合蛋白在体外具有显著的促进PBMC增殖的作用。结论:GST-IL-21融合蛋白在原核表达系统中可以有效表达,并具有较好的生物学活性。     

真核表达猪白细胞介素17及其生物活性研究

目的:为真核表达猪白细胞介素17(IL-17),研究产物在细胞培养下的免疫生物活性。方法:通过PCR扩增出猪IL-17基因并插入到真核表达载体p VAX1,然后转染到IPEC-J2细胞、Ha Ca T细胞和L02细胞中。在转染后第24、48和72h收集细胞,第48h收集上清液。收集细胞通过实时荧光定量PCR检测相关免疫基因的表达水平,收集上清液通过抑菌试验检测相关抗菌肽的生物活性。结果:采用p VAX1载体构建了表达猪IL-17的重组质粒,转染到细胞中。证实IL-17基因能诱导抗菌肽基因(RegⅢ、S100A8和BD2)的表达,显著上调JAK-STAT信号通路基因(JAK1、STAT1和STAT3)和细胞因子基因(IL-6、IL-12和TNF-α)的表达。此外,细胞上清液能够在不同程度上抑制大肠杆菌及金黄色葡萄球菌的增殖。结论:成功将猪IL-17基因真核表达,其表达产物能诱导效应细胞表达多种细胞因子,产生多种抗菌肽,具有抑菌能力;这为进一步研发猪IL-17作为抗菌免疫分子制剂奠定了初步基础。     

树鼩白细胞介素6的原核表达、纯化及免疫原性鉴定

目的:克隆树鼩白细胞介素6(IL-6)基因,在大肠杆菌中高效表达、纯化,并鉴定免疫原性。方法:根据GenBank预测的树鼩IL-6基因序列设计引物并扩增基因,克隆至原核表达载体pET-30a(+),构建的重组表达质粒pET-30a(+)-IL-6转化大肠杆菌BL21,IPTG诱导表达;表达的融合蛋白经Ni柱纯化后,Western印迹和ELISA检测其免疫原性,并检测不同组织中IL-6的分布情况。结果:酶切及测序证实重组表达质粒pET-30a(+)-IL-6构建正确;表达的重组蛋白相对分子质量约为29×10~3,纯化后复性的重组蛋白纯度可达95%以上,免疫兔子后Western印迹和ELISA鉴定其具有免疫原性,在气管、心、脾、肾中能检测到IL-6。结论:在大肠杆菌中高效表达了重组树鼩IL-6,纯化复性后具有良好的免疫原性。     

pLNCX-pemt2重组载体的构建ptpemt2基因在大鼠肝癌CBRH-7919细胞中的表达

为进一步研究pemt2对肝癌细胞生长抑制的作用机制提供方便的实验模型,构建了pLNCX-pemt2重组体.将目的基因pemt2连接入含有neo抗性基因的真核细胞表达载体pLNCX中,构建pLNCX-pemt2重组子,并用磷酸钙沉淀法将其转入大鼠肝癌CBRH-7919细胞中,应用PCR、Western印迹及[3H]SAM参入等技术对其转染、表达及活性进行鉴定.转染pLNCX-pemt2的大鼠肝癌细胞,PEMT2成功表达(分子量为22.5kD);高表达克隆PEMT2的表达量对照组高约5倍,其活性比对照组高2.1倍;细胞生长的倍增时间从21.54±7.08h延长到43.22±7.11h.结果表明,pLNCX-pemt2重组体转入肝癌细胞后,PEMT2蛋白得到高效表达,明显抑制肝癌细胞生长.     

High level expression and purification of active recombinant human interleukin-8 in Pichia pastoris

Human interleukin-8 (hIL-8) is a member of interleukin family which functions as a chemotactic factor as well as an angiogenesis mediator. Previously, a study reported that hIL-8 could be purified from inclusion bodies using a prokaryotic expression system, however, the required re-naturation step limits the recovery of fully active protein. In this study, soluble recombinant hIL-8 was expressed as a secreted protein at high level in Pichia pastoris under the control of AOX1 (alcohol oxidase 1) promoter. A simple purification strategy was established to recover rhIL-8 from the fermentation supernatant. The process includes precipitation with 80% saturation ammonium sulfate and CM Sepharose ion exchange chromatography, yielding 30 mg/L purified rhIL-8 at over 95% purity. The obtained rhIL-8 displays high specific activity, stimulating the migration of mouse neutrophils at concentrations as low as 0.25 ng/mL. Our results demonstrate that P. pastoris expression system is an efficient tool for large-scale manufacture of active recombinant hIL-8 for various applications.     

Expression, Identification and Characterize of CD25-Binding Epitope Modified Human IL-2 in Pichia pastoris

Interleukin-2 (IL-2) plays important roles in variety of immune functions and it is widely used in the medication. But in recent years it was reported that vascular leak syndrome (VLS) was induced by IL-2. Evidences showed that the interaction of IL-2 and IL-2Rαβγ (CD25) caused VLS. Thus, this experiment modified the CD25-binding epitope in human IL-2 (hIL-2) to minimize the side effect of IL-2 in the medication. In this study, a recombinant human interleukin 2 (rhIL-2) was expressed in Pichia (P.) pastoris. An effective strategy was established to express rhIL-2 protein in 120 L scale and the optimal purification procedure was investigated. The purity of rhIL-2 in final product was about 98 % and the concentration of the rhIL-2 was 0.45 mg/mL. Bioactivity analysis showed that the purified rhIL-2 protein displayed high activity on proliferation of CTLL-2 cells and increased the ratio of CD4⁺/CD8⁺. It indicates that the target protein is expressed and the character of the rhIL-2 has high activity. This study provides a strategy for large-scale production of bioactive rhIL-2 protein using P. pastoris as an expression host.     

Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni–NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.     

A Hybrid System Using Both Promoter Activation and Gene Amplification for Establishing Exogenous Protein Hyper-Producing Cell Lines

We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyper-producing cell line, D29, which exhibited a peak productivity rate of approximately 40 μg hIL-6 10^?6 cells day^?1 through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 μg hIL-6 ml^?1 per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.     

Human, viral or mutant human IL-10 expressed after local adenovirus-mediated gene transfer are equally effective in ameliorating disease pathology in a rabbit knee model of antigen-induced arthritis

IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral responses. Human IL-10 (hIL-10) has anti-inflammatory, immunosuppressive as well as immunostimulatory characteristics, whereas viral IL-10 (vIL-10), a homologue of hIL-10 encoded by Epstein Barr virus (EBV), lacks several immunostimulatory functions. The immunostimulatory characteristic of hIL-10 has been attributed to a single amino acid, isoleucine at position 87, which in vIL-10 is alanine. A mutant hIL-10 in which isoleucine has been substituted (mut.hIL-10) is biologically active with only immunosuppressive, but not immunostimulatory, functions, making it a potentially superior therapeutic for inflammatory diseases. To compare the efficacy of mut.hIL-10 with hIL-10 and vIL-10 in blocking the progression of rheumatoid arthritis, we used replication defective adenoviral vectors to deliver intra-articularly the gene encoding hIL-10, vIL-10 or mut.hIL-10 to antigen-induced arthritic (AIA) knee joints in rabbits. Intra-articular expression of hIL-10, vIL-10, and mut.hIL-10 resulted in significant improvement of the pathology in the treated joints to similar levels. These observed changes included a significant reduction in intra-articular leukocytosis and the degree of synovitis, as well as normalization of cartilage matrix metabolism. Our results suggest that hIL-10, vIL-10, and mut.hIL-10 are all equally therapeutic in the rabbit AIA model for treating disease pathology.     

Expression of a bioactive recombinant human interleukin-11 in chicken HD11 cell line

To direct the synthesis and secretion of recombinant human interleukin-11 (rhIL-11) in chicken HD11 cells, a plasmid targeting the c-lysozyme gene has been constructed which contains the mature cytokine cDNA in frame with the lysozyme leader sequence. The upregulation of rhIL-11 mediated by LPS proves the knock-in of hIL-11 cDNA in the lysozyme gene. The bioactivity of the expressed protein is demonstrated and quantified with the hIL-11 dependent 7TD1 and B9 cell lines. The electrophoretic mobility, receptor binding properties and growth promoting effect of the chicken-derived cytokine are identical to those of a rhIL-11 expressed in Escherichia coli. These results describe the secretion of a biologically active rhIL-11 expressed by an avian cellular machinery.     

人IL-17F对裸鼠人肝癌移植瘤生长的影响

利用RT-PCR方法从PHA活化的人外周血单个核细胞(PBMCs)中克隆hIL-17F基因,亚克隆至逆转录病毒载体pSIV-1,与辅助病毒载体pHIT456和pHIT60脂质体法共转染293T包装细胞,获得的成熟重组逆转录病毒(RV-hIL-17F)再感染SMMC-7721人肝癌细胞,并经G418筛选建立hIL-17F转基因肝癌细胞。PCR、RT-PCR和Westernblot结果表明hIL-17F基因在肝癌细胞中能成功整合、转录和表达。MTT和FCM结果表明hIL-17F不能改变SMMC-7721肝癌细胞的增殖活力和细胞周期,但ELISA结果表明其能明显下调肝癌细胞IL-6、IL-8和VEGF的表达。转基因肝癌细胞rhIL-17F表达上清具有抑制ECV304人脐静脉内皮细胞生长的作用。裸鼠皮下成瘤试验结果表明hIL-17F转基因肝癌细胞裸鼠致瘤能力明显减弱,VEGF和CD34表达降低,血管形成显著减少。hIL-17F可通过减少肿瘤血管形成显著抑制裸鼠人肝癌移植瘤的生长,为其进一步开展肿瘤血管靶向基因治疗和开发抗血管新药提供了一定的实验依据。     

Signal transduction by human herpesvirus 8 viral interleukin-6 (vIL-6) is modulated by the nonsignaling gp80 subunit of the IL-6 receptor complex and is distinct from signaling induced by human IL-6

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) mediates signaling through the gp130 signal transducer but unlike human IL-6 (hIL-6) does not require the nonsignaling gp80 alpha subunit of the IL-6 receptor complex. By utilizing a gp80-refractory vIL-6 variant, vIL-6(R189L), we found that signal transduction, as measured by STAT1 and STAT3 activation and gp130 tyrosine phosphorylation in gp80+/gp130+ HEK293T cells, was modulated by gp80. Furthermore, the signaling and BAF-130 cell growth-promoting activities of vIL-6 and hIL-6 could be distinguished, and exogenous addition of soluble gp80 enhanced cell growth supported by vIL-6. Our findings demonstrate that gp80 can modulate vIL-6 activity and that vIL-6 and hIL-6 signaling are not directly equivalent.