稳定表达小鼠CD40L的NIH3T3细胞系的建立及其在B细胞培养和激活中的应用

该研究构建小鼠CD40L真核表达重组质粒pcDNA3.1-mCD40L,通过电转法将重组质粒转至NIH3T3细胞中。利用G418对转染后细胞进行压力筛选,获得稳定转染细胞株。提取稳定转染细胞株RNA,通过RT-PCR法检测Neo基因的mRNA表达情况。分离稳定转染细胞上清,利用ELISA法检测小鼠CD40L蛋白水平的表达情况。RT-PCR结果显示,Neo基因能够在稳定转染细胞中表达,ELISA结果显示,获得的稳定转染细胞株NIH3T3-mCD40L细胞上清中CD40L的表达量高达1.286 ng/mL。进一步活性研究表明,该细胞系能够在体外与IL-2和IL-21共同作用培养B细胞至14天,并刺激B细胞产生特异性抗体。该细胞系的成功构建,为利用体外B细胞分离培养和活化法分离特异性单克隆抗体奠定了良好的基础。     

小菜蛾普通气味结合蛋白2基因的原核表达与多克隆抗体制备

本实验提取羽化3 d的小菜蛾Plutella xylostella成虫触角总RNA,反转录合成cDNA,以此为模板PCR扩增出小菜蛾普通气味结合蛋白2基因,大小为492 bp,Blast结果显示与多种昆虫的GOBP2具有较高的同源性.将该基因克隆到表达载体pMAL-c4E中,转化宿主菌TB1(DE3),获得单克隆重组质粒pMAL-c4E-GOBP2.IPTG成功诱导pMAL-c4E-GOBP2表达出约60 kDa的融合蛋白.优化诱导条件为3 mmol/L终浓度IPTG、6 h,可获得大量可溶性蛋白.表达的融合蛋白通过亲和色谱法纯化、免疫新西兰大白兔制备多克隆抗体.ELISA分析表明制备的抗体效价达1:1.28×105.     

不同信号肽对人鼠嵌合CMV-IgM分泌表达的影响

为了实现在体外制备巨细胞病毒-免疫球蛋白M(CMV-IgM)和探讨不同信号肽对CMV-IgM表达的影响,通过RLM-RACE技术钓取杂交瘤细胞基因序列,构建人鼠嵌合CMV-IgM表达载体,并通过PCR方法将5种不同的分泌型信号肽(SigA–SigE)代替CMV-IgM自身信号肽(SigF),利用中国仓鼠卵巢细胞(CHO)作为宿主细胞进行体外表达。对纯化后的CMV-IgM进行SDS-PAGE、SEC-HPLC和酶联免疫吸附试验(ELISA)确定其蛋白表达水平与免疫反应性,最终成功制备出910 kDa的重组蛋白,且研究表明,5种信号肽(SigA–SigE)的人鼠嵌合CMV-IgM表达量较CMV 6#细胞株自身信号肽SigF相比,分别提高了6.72、5.19、1.44、1.85、1.98倍。这将对人鼠嵌合CMV-IgM的开发提供理论基础,且为提高CMV-IgM表达量的研究工作提供了新思路。     

抗CD20抗体高产细胞株的筛选及质量评估

目的:筛选高表达单克隆细胞株,并通过优化培养基及流加物,最终达到提高目的蛋白产量及质量的目的。方法:通过有限稀释法对转染目的蛋白的CHO-S细胞进行单克隆化,应用双抗夹心ELISA方法对单克隆细胞株抗体表达量进行初步评估,最后根据筛选细胞株的活率、密度、产量及代谢情况,选择2~3株单克隆细胞进行培养条件优化,并对获得的发酵液进行纯化捕获,根据抗体蛋白表达量、糖型、等电点、纯度、酸碱峰分布等进行相应的评估分析,筛选出最优细胞株及最优培养方案。结果:经过单克隆化处理以及培养条件优化,蛋白的表达量由初始的不到500mg/L提升到2 290mg/L,且抗体蛋白纯度高达97.48%。抗体蛋白质量分析结果显示B1方案为该实验最优培养方案。结论:通过细胞株筛选、培养基优化能显著提高抗体蛋白的产量及质量,同时对抗体蛋白糖型、等电点、纯度等均有一定程度的优化。因此工业生产中可以通过高表达克隆的筛选、培养工艺优化等对目的蛋白产量及质量进行一定程度的改善与提高,对后期实验研究及工业化方案开发都具有很好的指导意义。     

菜蛾盘绒茧蜂多分DNA病毒EP-1-like基因克隆、原核表达与多克隆抗体制备方法

本实验提取被菜蛾盘绒茧蜂寄生后24h的小菜蛾幼虫体内总RNA,反转录合成cDNA,以此为模板PCR扩增出菜蛾盘绒茧蜂多分DNA病毒（Cotesia vestalis polydnavirus,CvBV）EP-1-like基因,将其分别克隆到表达载体pET30a、pET28a中,转化宿主菌BL21（DE3）,获得单克隆重组质粒pET-30a-EP1L和pET-28a-EP1L。经IPTG诱导,pET-28a-EP1L成功表达了约34.8kDa的包涵体蛋白。将诱导条件优化以后,采用割胶回收的方法纯化包涵体,将纯化后的表达蛋白免疫新西兰大耳兔制备多克隆抗体。ELISA分析表明制备的抗体效价达1：128000,Western blot检测证明抗体具有良好的免疫反应特异性。     

人IL35-IgG4(Fc)融合蛋白在CHO/DG44细胞中的稳定表达

本研究利用基因重组技术构建人IL35-IgG4(Fc)融合基因真核表达载体, 稳定转染CHO/DG44细胞并检测重组蛋白的表达。主要采用聚合酶链式反应(PCR)从脂多糖(Lipopolysaccharides, LPS)诱导的人髓性白血病细胞株KG-I cDNA文库中克隆EBI3和IL-12p35 cDNA, 重叠PCR法连接2个片段, 并克隆到IgG4(Fc)- pOptiVEC?-TOPO?载体上,对新构建的IL-35-IgG4 (Fc) pOptiVEC?-TOPO?真核表达载体并进行酶切、测序、PCR鉴定; 脂质体法转染CHO/DG44细胞; RT-PCR检测转染结果, 采用a-MEM-培养基筛选实验组细胞, 对筛选的阳性克隆细胞再进行氨甲喋呤(Methotrexate, MTX)的加压筛选, ProteinG-Agarose纯化阳性克隆培养上清, 免疫印迹检测目的蛋白表达。结果显示IL-35-IgG4 (Fc) pOptiVEC?-TOPO?表达载体稳定转染CHO/DG44细胞并获得阳性克隆; SDS-PAGE电泳得到一条与预期相对分子质量大小相符的蛋白条带; 该蛋白能与羊抗人IgG4抗体特异结合。本实验获得了能够稳定表达具有稳定结构的IL35-IgG4(Fc)融合蛋白的CHO/DG44细胞株。     

一种具有人VEGF结合活性的人源单一重链可变区的克隆与表达

为避免一种来自五特征转基因小鼠的全人VEGF单克隆IgM抗体分子量大的不足,本研究探讨了该抗体单一重链可变区的功能特性。首先,PCR获得该抗体的重链可变区,将该序列克隆至pET28a表达载体内,在大肠杆菌中进行了诱导表达。通过变性纯化和复性等方法获得了具有生物学活性的16kDa重组抗体片段——rhVVH。体外结合实验表明,rhVVH保留有完整免疫球蛋白的人VEGF结合活性。人脐静脉内皮细胞(HUVEC)增殖抑制实验表明:rhVVH可以剂量依赖性的抑制HUVEC的增殖。上述结果揭示了该抗体单一重链可变区保留有完整抗体的部分功能,为进一步开展全人源VEGF单克隆IgM抗体小型化研究奠定了基础。     

人ABCB6表达载体的构建及稳定表达细胞株A375的筛选

目的构建表达载体pIRES2-ZsGreen1-ABCB6,在转染的人黑素瘤细胞系株A375中筛选其稳定表达的细胞株。方法抽取健康人外周血,分离外周血单个核细胞,提取总RNA,逆转录获取cDNA序列,加入特异性引物经PCR扩增获得ABCB6cDNA双链,再经过BglII、EcoRI双酶切PCR产物及质粒载体pIRES2-ZsGreen1,酶切产物经回收、T4DNA连接酶连接,产物转化到大肠杆菌DH5α,挑取阳性克隆经菌落PCR鉴定、酶切鉴定和测序分析,以确定构建质粒正确。转染人黑素瘤细胞株A375,G418筛选稳定表达ABCB6的单克隆细胞株,应用荧光显微镜鉴定ABCB6蛋白的表达情况。结果 pIRES2-ZsGreen1-ABCB6质粒经菌落PCR、酶切、测序鉴定正确,经过G418筛选后获得稳定细胞株,在荧光显微镜下可观察到绿色荧光蛋白在A375细胞中的表达。结论表达载体pIRES2-ZsGreen1-ABCB6构建正确,并成功筛选出稳定表达ABCB6的A375细胞株,为进一步研究ABCB6的生物学功能奠定了良好基础。     

短发夹RNA载体稳定抑制端粒相关蛋白TERF2IP1表达的HCT116细胞的构建

目的:构建针对TERF2IP1基因的短发夹RNA(sh RNA)表达载体,在HCT116细胞中鉴定该载体对TERF2IP1基因的干扰效果。方法:设计针对TERF2IP1基因的sh RNA序列,将其克隆至p GPHI/Hygro载体中,瞬时转染HCT116细胞48 h后,用400 mg/m L的潮霉素筛选抗性克隆,获得阳性单克隆细胞株;分别提取细胞总RNA和蛋白,进行RT-PCR和Western印迹,检测TERF2IP1的表达水平。结果:构建了4对针对TERF2IP1基因的sh RNA表达载体,通过潮霉素筛选获得稳定干扰TERF2IP1基因的HCT116细胞系,实时定量PCR和Western印迹实验均证实潮霉素筛选后,HCT116细胞中TERF2IP1的表达水平明显降低。结论:构建了4对人TERF2IP1基因的sh RNA表达载体,获得4株分别针对TERF2IP1基因不同位点的稳定干扰HCT116细胞株。     

黄颡鱼免疫球蛋白M重链基因原核表达研究

在前期研究克隆得到黄颡鱼(Pelteobagrus fulvidraco)全长cDNA的基础上, 为进一步研究黄颡鱼免疫球蛋白M(IgM)重链的生物学功能, 设计特异性引物PCR扩增获得了编码成熟免疫球蛋白M重链基因的编码序列。将该基因编码片段连接到原核表达载体pQE30中, 构建黄颡鱼IgM重链的重组表达质粒IgM-pQE30。该重组质粒经酶切和测序鉴定后, 转入表达宿主大肠杆菌M15中诱导表达, 在30℃下, 经0.2 mmol/L IPTG诱导表达8 h, 可获得大量以包涵体形式存在的黄颡鱼IgM重链蛋白, 经SDS-PAGE电泳和Western blotting 分析表明, 新增的62 kDa蛋白条带与预期值相符, 且能与鼠源抗6×His的单克隆抗体特异性结合, 证明黄颡鱼IgM重链基因获得了高效原核表达。为进一步纯化该蛋白制备特异抗体, 研究其生物学功能奠定了基础。     

Selective macrophage activation by muramyldipeptide bound to monoclonal antibodies specific for mouse tumor cells

Summary IgM monoclonal antibodies directed against tumor cells which do not mediate antibody-dependent macrophage cytotoxicity (ADMC) even when they are cytotoxic in the presence of complement, have been shown to render macrophages tumoricidal when they carry an immunomodulating agent, i.e., muramyldipeptide (MDP).This statement is based on experiments using two IgM monoclonal antibodies selected for their ability to bind L1210 leukemia cells (F₂-10-23-IgM) and 3LL Lewis lung carcinoma cells (6B6-IgM) specifically, as shown by flow cytofluorometry analysis.The MDP-IgM conjugates, containing 45 MDP molecules per IgM molecule, were prepared by allowing MDP-hydroxy-succinimide ester to react with IgM monoclonal antibodies.The MDP-IgM conjugates are shown to bind to relevant tumor cells and to induce the activation of thioglycolate-elicited peritoneal mouse macrophages leading to 80% growth inhibition of target cells at optimum concentrations of bound MDP. These concentrations of bound MDP were 10 times lower than the concentration of free MDP, giving a maximum activation that is limited to 20% growth inhibition.No macrophage activation was evidenced when tumor cells were incubated in the presence of irrelevant MDP-IgM conjugates and macrophages or when macrophages were preincubated in the presence of MDP-IgM conjugates and then incubated in the presence of relevant or irrelevant tumor cells but in the absence of the MDP-IgM conjugates.The reported results are discussed with reference to the mechanism of activation of macrophage by muramyldipeptide and to the usefulness of such MDP-IgM conjugates as potential antitumor agents in cancer therapy.Abbreviations ADMC antibody dependent macrophage mediated cytotoxicity - F-GAM fluoresceinylthiocarbamyl goat anti mouse antibody - -Man-BSA -mannopyranosyl-phenylthiocarbamyl bovine serum albumin containing some 25 mannose residues (neoglycoprotein) - MDP muramyldipeptide, 2-acetamido-3(2-0-d-lactyl-l-alanyl-d-glutamyl amine) glucopyranose - MDP-F₂-10-23-IgM Murine monoclonal antibody specific of L1210 leukemia cells and substituted with 45 MDP molecules - MDP—6B6-IgM Murine monoclonal antibody specific of 3LL Lewis lung carcinoma cells and substituted with 45 MDP molecules - MEM minimal essential medium - TDM Trehalose-dimycolate     

B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies

The recent development of screening strategies based on the generation and display of large libraries of antibody fragments has allowed considerable advances for the in vitro isolation of monoclonal antibodies (mAbs). We previously developed a technology referred to as the 'ADLib (Autonomously Diversifying Library) system', which allows the rapid screening and isolation in vitro of antigen-specific monoclonal antibodies (mAbs) from libraries of immunoglobulin M (IgM) displayed by the chicken B-cell line DT40. Here, we report a novel application of the ADLib system to the production of chimeric human mAbs. We have designed gene knock-in constructs to generate DT40 strains that coexpress chimeric human IgG and chicken IgM via B-cell-specific RNA alternative splicing. We demonstrate that the application of the ADLib system to these strains allows the one-step selection of antigen-specific human chimeric IgG. In addition, the production of chimeric IgG can be selectively increased when we modulate RNA processing by overexpressing the polyadenylation factor CstF-64. This method provides a new way to efficiently design mAbs suitable for a wide range of purposes including antibody therapy.     

鲈鱼IgM重链基因cDNA的克隆及其原核表达

采用同源克隆和RACE技术扩增到鲈鱼 (Lateolabrax japonicus) 免疫球蛋白M (Immunoglobulin M,IgM) 重链 (Heavy chain,H) 基因全长cDNA序列。鲈鱼IgM cDNA全长为1 901 bp,开放阅读框包含1 749 bp,编码582个氨基酸。根据鲈鱼IgM和其他硬骨鱼免疫球蛋白重链恒定区的氨基酸序列构建的系统发育树表明IgM、IgZ和IgD分别聚为一枝,其中IgM与IgZ分支的进化关系较近,而与IgD分支的进化关系较远。RT-PCR检测IgM在鲈鱼各组织器官的表达情况,其中在头肾及脾脏中表达量最高,心脏、肌肉及脑中几乎不表达。利用已获得的鲈鱼IgM cDNA序列,构建原核表达载体pQE30-IgM,并在M15大肠杆菌中成功诱导表达了分子量为63kD的重组蛋白His-IgM,Western-blotting显示鲈鱼IgM重组蛋白能与鼠源抗6×His的单克隆抗体特异性结合,说明已经获得了基因工程表达IgM重链蛋白。     

抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化

目的：FGF2是肿瘤血管新生过程中最重要的因子之一,因此通过制备抗FGF2人鼠嵌合抗体中和其发挥作用,以达到抑制肿瘤生长的目的。方法：利用分泌抗FGF2抗体的杂交瘤细胞株IgG9B9和人B淋巴细胞,分别克隆抗体轻链可变区V_L、重链可变区V_H和人重链恒定区C_H基因,从pComb3λ载体中扩增出人λ 链恒定区C_L基因,通过重叠PCR,将V_L,V_H和C_L,C_H片段分别连接形成嵌合抗体的轻链L和重链H,将L/H链以单独构建或串联于同一载体的方式,构建抗FGF2嵌合抗体表达载体,并通过调控元件WPRE优化载体、共转染促生长因子aFGF以及调整表达温度等方式提高嵌合抗体在真核细胞中的表达。结果：成功构建了优化表达载体PLexm-WPRE、PLexm-aFGF;L、H链基因也成功构建,并以L、H或L-F2A-H（2A连接肽将L和H连接起来）的方式分别成功连接到PLexm,PLexm-WPRE载体中。转染细胞上清的ELISA鉴定结果表明,L/H链单独构建要比串联构建的方式具有更高的表达水平,WPRE能有效促进抗体的表达而aFGF并不能促进其表达,与31、37℃相比,33℃时抗体的表达量最高,同时嵌合抗体表现出了很好的结合活性及中和活性,竞争IC50=6.25μg/ml。通过亲和层析获得了高纯度的抗FGF2嵌合抗体。结论：在33℃下,人鼠嵌合抗体基因在WPRE存在下表达量最高,且与抗原FGF2有很好的结合活性及中和活性,为临床应用奠定了基础。     

Localization of a Passively Transferred Human Recombinant Monoclonal Antibody to Herpes Simplex Virus Glycoprotein D to Infected Nerve Fibers and Sensory Neurons In Vivo

A human recombinant monoclonal antibody to herpes simplex virus (HSV) glycoprotein D labeled with the fluorescent dye Cy5 was administered to mice infected in the cornea with HSV type 1 (HSV-1). The distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. The antibody was detected on HSV-infected nerve fibers in the cornea--identified by colocalization with HSV antigens and the neuritic markers neurofilament, GAP-43, synapsin-1, and CNPase--and on the perikarya of sensory neurons in the HSV-1-infected neurons in ipsilateral trigeminal ganglia. Antibodies have been shown to be effective against many neurotropic viruses, often in the absence of obvious cell damage. Observations from experimental HSV infections suggest that antibodies could act in part by interfering with virus expression in the ganglia and/or with axonal spread. The present results provide morphological evidence of the localization of antiviral antibodies at anatomical sites relevant to such putative antibody-mediated protective actions and suggest that viral glycoproteins are accessible to antibodies on infected nerve fibers and sensory neurons.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Competitive inhibition by human sera of mouse monoclonal antibody binding to glycoproteins C and D of herpes simplex virus types 1 and 2.

A competitive enzyme-linked immunosorbent assay was used to test for human antibodies to antigenic sites on herpes simplex virus (HSV) glycoproteins C and D, which are recognized by mouse monoclonal antibodies. Antibodies capable of blocking the monoclonal antibodies were detected in the human sera, and the inhibition of binding correlated with the histories of herpetic infections. The binding of monoclonal antibody to glycoprotein C of HSV type 2 was inhibited primarily by sera from patients with recurrent herpes genitalis; however, the binding of the monoclonal antibodies to gC of HSV type 1 was inhibited by sera from patients previously infected with either HSV type 1 or HSV type 2. The observations suggest that the antigenic sites defined by the mouse monoclonal antibodies are recognized by the human host.     

Molecular Analysis of the Gal/GalNAc Adhesin of Entamoeba histolytica1

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactosc/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E. coli, retained at least some of its native conformation.