苦荞黄酮-3-羟化酶截短体的原核表达与多克隆抗体制备

为了制备苦荞黄酮-3-羟化酶的多克隆抗体,该研究以苦荞种子灌浆期cDNA文库中获得的苦荞黄酮-3-羟化酶基因截短体(truncated Flavanone-3-hydroxylase,TrF3 H)序列为基础,采用PCR扩增F3 H的截短序列编码区(TrF3 H),构建了原核表达载体pET47b-TrF3 H,并转化入大肠杆菌Rosetta(DE3)plysS中进行诱导表达,将经钴离子螯合层析柱纯化后的目的蛋白切胶回收后制备了高效价的多克隆抗体。结果表明:pET47b-TrF3 H在大肠杆菌Rosetta(DE3)plysS中以包涵体的形式高效表达。蛋白质印迹显示,制备的多克隆抗体能特异识别其对应的抗原,天然的黄酮-3-羟化酶蛋白在苦荞的未成熟种子中大量表达。原核表达体系的建立和多克隆抗体的制备为进一步探讨F3H在苦荞中功能奠定了基础。     

Electro-immunoblotting characterization ofPseudo-nitzschia multiseries andP. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence     

二点委夜蛾非典型嗅觉受体AlepOrco的基因克隆、原核表达及多克隆抗体制备

【目的】非典型嗅觉受体(olfactory receptor co-receptor, Orco)与典型嗅觉受体共同形成离子通道,在昆虫嗅觉识别中具有至关重要的作用。本研究旨在克隆和表达二点委夜蛾Athetis lepigone Orco基因,明确其分子特性,为进一步研究该基因在二点委夜蛾中的功能奠定基础。【方法】将二点委夜蛾雌雄成虫触角转录组数据建立本地数据库,通过生物信息学分析获得二点委夜蛾Orco同源基因AlepOrco;利用RT-PCR方法克隆二点委夜蛾AlepOrco基因全长,并在pGEX-6P-1/BL21(DE3)系统中进行了该基因开放阅读框(ORF)的原核表达,制备多克隆抗体,用Western blot检测抗体特异性;利用qPCR技术检测该基因在二点委夜蛾雌雄成虫不同组织(喙、触角、去除触角和喙的头、胸、腹、足和翅)中的表达谱。【结果】获得了二点委夜蛾AlepOrco的cDNA(GenBank登录号:MN583125)全长序列,开放阅读框长1 422 bp,编码473个氨基酸,序列中有7个跨膜结构区,预测等电点为8.59,分子量为53.40 kD。SDS-PAGE和We...     

Special Features of the DNA-Hydrolyzing Activity of the Antibodies in Systemic Lupus Erythematosus

Two types of IgG anti-DNA antibodies exhibiting DNA-hydrolyzing activity have been isolated from blood serum of patients with systemic lupus erythematosus. This DNase activity of antibodies differs from serum DNases by the non-processive mode, temperature resistance, pH optimum, and the rate of DNA hydrolysis. It is suggested that the anti-DNA antibody molecule possessing DNase activity contains two sites: one site determines specificity of antibody-DNA interaction, whereas the other is responsible for manifestation of the catalytic activity.     

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

斑马鱼基因ATF4多克隆抗体的制备

ATF4是含有bZIP结构域的ATF／CREB转录因子家族成员,对胚胎的发育以及细胞的增殖、分化有重要的调节作用。制备ATF4的多克隆抗体对于研究其在斑马鱼心脏发育过程中的作用有重要的意义。研究首先通过生物信息学方法,选择ATF4基因中特异性强、具亲水性的一段核苷酸序列（1017bp）,通过PCR扩增,将片段重组到原核表达载体pET-28a,然后转化入Rosetta菌株中。经测序鉴定正确后,用IPTG诱导表达融合蛋白,以该融合蛋白免疫小鼠,获得ATF4多克隆抗鼠血清。对该多抗血清抗体进行验证,具有很好特异性和较高效价,可以用作Western—blotting、免疫印迹等试验分析。     

水稻YTB中OSVDAC5蛋白的原核表达与抗体制备

目的:克隆水稻YTB osvdac5基因,原核表达后获得纯化的OSVDAC5蛋白,制备相应的抗体.方法:采用Trizol法提取水稻总mRNA,反转录为cDNA,通过PCR扩增得到该基因与原核表达载体连接,构建重组质粒pET-30a-osvdac5,并转入大肠杆菌进行原核表达,SDS-PAGE检测表达产物.通过镍柱纯化获得的单一目的蛋白用于抗体制备,用Western Blot检测抗体的特异性.结果:克隆到原核表达载体中osvdac5基因的ORF为813 bp,编码271个氨基酸.在大肠杆菌中15℃、0.7mmol/L的IPTG浓度诱导17 h是pET-30a-osvdac5融合蛋白表达的优选条件,表达的OSVDAC5蛋白属于包涵体蛋白.镍柱纯化后的OSVDAC5为30 kD左右的单一条带.Western Blot分析表明,抗体能够与30 kD处的OSVDAC5蛋白进行特异性结合.结论:成功克隆了水稻YTB osvdac5基因,原核表达蛋白OSVDAC5制备的多免隆抗体具有一定特异性,能与免疫抗原结合,这为进一步研究OSVDAC5蛋白在植物不同生长发育时期中的表达模式奠定了基础.     

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 .     

Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins

The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre‐depleted or post‐depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber‐flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.