人SUMO-3基因原核表达及多克隆抗体制备

构建人SUMO-3基因的原核表达载体pET41a(+)-SUMO-3,表达重组GST-SUMO-3融合蛋白,制备人SUMO-3多克隆抗体。试验结果显示,通过PCR方法从重组质粒pEYFP-SUMO-3中克隆到的SUMO-3 N端93个氨基酸的基因序列与NCBI上提供的序列一致,重组质粒pET41a(+)-SUMO-3构建成功;重组pET41a(+)-SUMO-3在E.coli.BL21 (DE3) pLysS中表达GST-SUMO-3融合蛋白,分子量为44.0 kDa,与预期分子量一致;采用亲和层析纯化融合蛋白GST-SUMO-3并免疫家兔,获得人SUMO-3抗体;Western blot 检测显示该抗体可以特异性识别SUMO-3,ELISA检测结果成阳性,抗体效价约为1: 20000。实验结果为进一步研究人SUMO-3及SUMO第二类家族的功能提供了有用工具。     

Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgeulc animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein （EGFP） transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.     

Conversion of CD95 (Fas) Type II into Type I signaling by sub-lethal doses of cycloheximide

CD95 (Fas/Apo-1)-mediated apoptosis was shown to occur through two distinct pathways. One involves a direct activation of caspase-3 by large amounts of caspase-8 generated at the DISC (Type I cells). The other is related to the cleavage of Bid by low concentration of caspase-8, leading to the release of cytochrome c from mitochondria and the activation of caspase-3 by the cytochrome c/APAF-1/caspase-9 apoptosome (Type II cells). It is also known that the protein synthesis inhibitor cycloheximide (CHX) sensitizes Type I cells to CD95-mediated apoptosis, but it remains contradictory whether this effect also occurs in Type II cells. Here, we show that sub-lethal doses of CHX render both Type I and Type II cells sensitive to the apoptogenic effect of anti-CD95 antibodies but not to chemotherapeutic drugs. Moreover, Bcl-2-positive Type II cells become strongly sensitive to CD95-mediated apoptosis by the addition of CHX to the cell culture. This is not the result of a restraint of the anti-apoptotic effect of Bcl-2 at the mitochondrial level since CHX-treated Type II cells still retain their resistance to chemotherapeutic drugs. Therefore, CHX treatment is granting the CD95-mediated pathway the ability to bypass the mitochondria requirement to apoptosis, much alike to what is observed in Type I cells.     

用免疫亲和层析法纯化萝卜 PHGPx 天然蛋白

萝卜磷脂氢谷胱甘肽过氧化物酶 (RsPHGPx) 是一个定位于线粒体的蛋白质 . 为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的 RsPHGPx. 用重组 RsPHGPx 蛋白免疫兔子获得了抗 RsPHGPx 的多克隆抗血清,以重组 RsPHGPx 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗 RsPHGPx 的抗体 . 将纯化好的抗体偶联到一个 N- 羟基琥珀酰亚胺 (NHS) 预先激活的琼脂糖柱子上,装配成一个以单特异性的抗 RsPHGPx 抗体为配体的免疫亲和层析柱 . 经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案 . 按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质 . 免疫印迹分析表明,该蛋白质被抗 RsPHGPx 的抗血清特异识别 . 酶活性分析表明,该蛋白质具有显著的 PHGPx 活性 . 这些结果表明,纯化到的特异蛋白质是萝卜的 RsPHGPx 天然蛋白 . 这是首个关于定位于植物细胞器的 PHGPx 蛋白纯化的报道 . 这一结果为准确测定 RsPHGPx 信号肽的切割位点奠定了基础,并将有助于对植物 PHGPx 的亚细胞定位机制及其生理功能的深入研究 .     

一种新的垂体分泌蛋白Mimecan原核表达、抗体制备及其在垂体瘤组织中的表达(英文)

Mimecan osteoglycin是一种分泌型蛋白质 ,目前其功能尚不明确 .从人垂体cDNA中克隆到OIF基因并构建成重组表达质粒pGEX 5X 2 mimecan ,将此重组表达质粒转化大肠杆菌BL2 1(DE3)后用IPTG诱导 ,成功表达了一种分子量约为 38kD融合蛋白 ,约占菌体总蛋白的 30 %～ 4 0 % .此融合蛋白经纯化分离后免疫新西兰大白兔以制备多克隆抗体 .用Western印迹法检测兔的抗血清 .结果显示 ,该多克隆抗体有较好的针对mimecan蛋白的专一性并且效价较高 ,可用于对mimecan的功能研究 .用此多克隆抗体检测到在某些种类的人垂体瘤组织中mimecan表达极高 ,提示可能存在新的垂体瘤类型 .     

GGNBP1抗体制备及小鼠组织表达谱分析

体外实验研究表明配子生成素结合蛋白1(GGNBP1)可能与GGN1相互作用形成睾丸特异性复合物,在精子生成过程中发挥作用.从小鼠睾丸总RNA中反转录扩增Ggnbpl全长cDNA,构建表达质粒,在大肠杆菌中表达GGNBP1,经聚丙烯酰胺凝胶纯化后免疫新西兰白兔,制备兔多抗血清.镍离子金属螯合柱纯化表达的GGNBP1蛋白,与NHS活化基团交联,制备GGNBP1抗体亲和层析柱,纯化GGNBP1多抗.在293FT细胞中瞬时表达Myc-GGNBP1融合蛋白,用于Mvc单抗验证GGNBP1抗体特异性,结果证明获得了特异性的GGNBP1抗体.分别制备小鼠脑、心、肺、肝、脾、肾、肌肉、卵巢、睾丸和子宫组织匀浆,用GGNBP1抗体进行Western印迹分析,结果仅在睾丸组织匀浆中检测到GGNBP1特异性条带,证明GGNBP1是睾丸特异性表达蛋白.     

转录因子CTCF的克隆、表达及其抗体制备

用RT-PCR方法从HeLa细胞总RNA中扩增转录因子CTCF的cDNA序列,克隆至pGEM-TEasy载体上。将CTCF蛋白N段的编码序列亚克隆至pET22b( )载体中,在大肠杆菌BL21(DE3)中诱导表达,并用镍柱进行亲和纯化。用纯化后的CTCFN段蛋白免疫家兔,获得CTCF的多克隆抗体,进而用于细胞内CTCF蛋白的免疫印迹检测。     

FMDV整联蛋白受体b1亚基的克隆及其配体黏附区多抗的制备

采用RT-PCR技术从牛气管组织扩增出2400 bp的b1基因, 回收纯化连入PGEM-T载体, 测序。用Expasy软件对b1基因的抗原性进行分析, 选取胞外区334~861 bp的配体结合区与6×His融合, 在大肠杆菌中大规模诱导表达, 并经Ni2+亲和柱层析纯化。通过SDS-PAGE鉴定后, 应用纯化蛋白免疫新西兰家兔, 获得效价在1:12 800以上的多抗, Western blotting鉴定表明此抗体可特异性的与表达的融合蛋白作用。     

Nmi及其变异体在大肠杆菌中表达、抗体制备及其细胞分布

 为了检测细胞内源性Nmi和变异体Nmi s的表达、亚细胞分布以及可能形成的信号转导蛋白复合物 ,成功构建并表达了异源Nmi和Nmi s 获得纯化异源蛋白 ,用以制备兔源抗Nmi(Nmi s)多克隆抗体 .Western印迹在多种细胞株HL 60 ,K562 ,2 93T细胞中检测到Nmi(Nmi s)高表达 .在不同的细胞系中 ,Nmi表现为不同的蛋白复合物迁移带 ;Nmi(Nmi s)在胞浆和胞核均存在 ,而核内荧光更强 .Nmi(Nmi s)通过与多种蛋白相互作用行使其生物学功能 ,在不同细胞中可能与不同的蛋白相互作用 ,发挥不同的功能 .     

Chemiluminescent Enzyme Immunoassay for Measuring Leptin

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1–1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.