卵黄球蛋白Y的制备及应用

1卵黄球蛋白的性质正常鸡卵黄球蛋白Y（]gV）的分子量约为18万道尔顿,由2条轻链（2L）和2条重链（2H）组成,分子量分别为6～7万道尔顿和2．2～3．0万道尔顿,等电点约为5．2。与一般哺乳动物IgG相比,1gY具有较强的耐热、耐酸、抗离子强度和一定的抗酶降解能力。在低于75℃条件下,I“具有良好的热稳定性。IgY制剂在4℃贮存5年或在室温贮存6个月,其活性仍     

卵黄免疫球蛋白IgY的提纯及应用研究进展

１IgY的性质鸡卵黄免疫球蛋白（IgY）是鸡卵黄中存在的主要免疫球蛋白，当用某种抗原免疫母鸡时，母鸡产生免疫反应，在卵黄成熟期，血液中的免疫球蛋白IgG选择性的转移到卵黄中，称卵黄免疫球蛋白IgY，IgY在子鸡孵育过程中和孵育后对子鸡起保护作用。IgY的性质与哺乳动物的IgG类似，其分子量约１８０kDa，由两条轻链和两条重链组成（２H＋２L），其中轻链（L）分子量约２２０００Da，重链分子量约６７０００Da，等电点接近５．２。含氮量为１４．８％。IgY的氨基酸组成与鸡血清IgG和牛IgG不同（如表１）。持良好。根据MakotoSh…     

卵黄抗体在动物疾病防治上的应用进展

卵黄抗体是当今生物科学研究领域的一项重大发现。母鸡以特定抗原免疫后,能产生相对应的特异性抗体,并将其有效地储存在卵黄中。卵黄中的免疫球蛋白G(IgG)称卵黄抗体,又名lgY,自Williams等(1962)发现卵黄抗体以来,IgY以其取材方便,制作简单,价格便宜等优点,广泛应用于动物疾病的诊断、预防和治疗。1IgY的性质IgY的性质与哺乳动物的IgG相似。正常鸡的IgY的分子量约为180KDa,由两条轻链(2L)和两条重链(2H)组成,分子量分别为60～70KDa和22～30KDa。其等电点约为5.2。IgY具有良好的耐热能力。在低于75℃条件下,IgY具有良好的热稳定性…     

卵黄抗体分离、提取和纯化方法研究进展

卵黄抗体即卵黄免疫球蛋白(Egg Yolk Im-munoglobulin,IgY),目前研究较多的是鸡卵黄抗体。母鸡接受免疫后,在卵黄成熟期,血液中的免疫球蛋白IgG选择性地转移到卵黄中形成IgY。IgY是一种7 S免疫球蛋白,分子量180kD,由两条重链(分子量67～70 kD)和两条轻链(分子量22～30 kD)组成,等电点接近5.2。IgY的结构虽与IgG相似,但其Fc段氨基酸组成与IgG相差很大,不结合类风湿因子,不与蛋白A、G以及哺乳     

卵黄抗体纯化和浓缩的研究进展

卵黄抗体(immunoglobulin of egg yolk,IgY)是禽卵黄中存在的免疫球蛋白,是由禽血液中的IgG转移至卵黄中产生的对子代具有保护性作用的抗体。IgY的分子质量约为180 ku,由2条轻链和2条重链组成,其中轻链分子质量为22～30 ku,重链分子质量为67～70 ku,等电点接近5.2。与IgG相比,IgY具有动     

猪SIgA分泌片的分离纯化及其多克隆抗体的制备

为制备抗分泌片多克隆抗体,采用分子筛凝胶层析、离子交换层析和硫酸铵盐析法从猪初乳中分离纯化分泌片和SIgA。SDS—PAGE鉴定表明,纯化的SIgA呈现分泌片、重链和轻链三条带,大小约为70ku;凝胶薄层扫描分析SIgA和分泌片纯度分别为90％和89％;琼脂扩散鉴定表明,SIgA和分泌片与抗分泌片单克隆抗体发生了特异反应。以纯化分泌片为抗原,制备抗分泌片多克隆抗体,琼脂扩散检测多抗的效价为1：32。高纯度分泌片的提取及高效价抗分泌片多抗的制备为粘膜负．疫研究奠定了物质基础。     

抗猪IgG单克隆抗体的制备与应用

采用辛酸一硫酸铵盐析和SuperdexTM-200 凝胶层析分离纯化猪血清IgG,免疫BALB/c小鼠,利用细胞融合技术获得8株稳定分泌抗猪IgG 单克隆抗体的杂交瘤细胞.Ig 亚类分析表明,所制备单克隆抗体的轻链亚类均为K,重链除6H3为IgG2b外,其余7株均属于IgG1.Western blot分析显示,5株单克隆抗体识别猪IgG轻链,3株单克隆抗体识别猪IgG重链.以蛋白G亲和层析纯化单克隆抗体7H1腹水,用改良过碘酸钠法进行辣根过氧化物酶(HRP)标记,结果显示,HRP 标记抗猪IgG单克隆抗体对纯化猪IgG的工作浓度达1:12 800.将HRP标记鼠抗猪IgG单克隆抗体与HRP标记羊抗猪IgG多克隆抗体应用于猪血清圆环病毒抗体检测,二者符合率为95.12%,其ELISA和Westem blot的工作浓度为1:2 000和1:10000,表明HRP标记抗猪IgG单克隆抗体具有高度的敏感性.本研究制备的酶标抗猪IgG单克隆抗体为猪病的免疫诊断试剂研究和猪细胞生物学研究提供了一种基础工具.     

鸡免疫球蛋白(GIgG)轻链的分离纯化及其抗血清的制备

经硫酸铵盐析、柱色谱分离得到纯化的鸡血清IgG。继用2-巯基乙醇还原、碘乙酰胶烷化拆开IgG的轻、重链,再经Sephadex G100凝胶过滤柱色谱分离得到IgG轻链。以IgG轻链免疫家兔制得兔抗鸡IgG轻链抗血清。     

鸡SIgA重链单克隆抗体的制备及新城疫病毒和禽流感病毒特异黏膜SIgA检测方法的建立

为建立快速检测禽类黏膜组织中SIgA含量的方法,本研究原核重组表达鸡SIgA重链片段蛋白,纯化后免疫BALB/c鼠.以鸡胆汁中分离纯化的SIgA作为检测抗原,常规单克隆技术筛选出1株能够稳定分泌抗鸡SIgA单克隆抗体(MAb)的IgG2a、κ型MAb.经ELISA和western blot分析,所获得的1株MAb亲和力高、特异性强;采用生物素标记纯化MAb腹水IgG为检测二抗,以禽流感-新城疫重组二联活载体疫苗免疫SPF鸡为模型,初步建立了新城疫病毒和H5亚型禽流感病毒特异的黏膜SIgA间接ELISA检测方法.本研究为开展特异性家禽黏膜免疫机制的研究提供了重要技术手段.     

鸡SIgA的提取鉴定及其抗体制备

实验采用饱和硫酸铵法从健康肉鸡胆囊收集的胆汁中粗提二聚体SIgA,继而透析粗提产物除盐,然后进行纤维素DEAE-52离子交换层析,使用紫外分光光度计测定收集液的蛋白浓度,对层析产物浓缩使其浓度≥2.0 mg/m l,最后使用SDS-PAGE进行鉴定,从电泳所得图像中可以看到清晰的两个条带,一个条带分子量在67~70 ku之间为重链,另一个条带在26~28 ku之间为轻链。进一步将纯化的鸡SIgA抗体免疫健康兔,制备得到兔抗鸡SIgA抗体。     

Preparation of heavy chain specific antisera to bovine IgA, IgM, IgG1 and IgG2

Goats, guinea pigs and rabbits were immunized with bovine IgM or with intact molecules, heavy chains, Fc portions or light chains of bovine IgG1 and IgG2. Rabbits and guinea pigs were immunized with bovine secretory IgA. Goats and guinea pigs produced heavy chain specific antisera to intact IgM whereas rabbits produced anti-light chain antibody and in one instance anti-alpha 2-macroglobulin antibody in addition to the anti-mu response. Goats and guinea pigs produced antisera to bovine IgG1 and IgG2 and their Fc portions that needed little absorption to render them monospecific for the heavy chain. In addition to antibody to the heavy chains, rabbits produced anti-light chain antibody when immunized with intact IgG1 or IgG2 molecules. These latter sera, and those produced by rabbits immunized with Fc portions of IgG1 or IgG2 required extensive absorption before they were monospecific for their respective heavy chains. Heavy chains were poor immunogens in all three species. Rabbits immunized with IgA produced both anti-alpha and anti-light chain antibodies while guinea pigs produced sera with antibody activity to the alpha chain only.     

Monoclonal antibodies against ovine IgA purified from lung lavage fluid

Ovine secretory IgA (sIgA) has been purified to relative homogeneity by ammonium sulphate precipitation (to 40 per cent w/v) of lung lavage fluid from 3- or 12-month-old lambs, followed by molecular sieve chromatography on a Sephacryl S300 matrix. Three peaks A, B and C with molecular sizes corresponding to 550,000, 400,000 and 165,000 respectively were eluted from the column. Immunoelectrophoresis, radial immunodiffusion and enzyme-linked immunosorbent assays (ELISAs) with class specific antiserum confirmed that peak B contained only IgA. Polyacrylamide gel electrophoresis of peak B under reducing conditions resolved three subunits corresponding to secretory component, heavy and light chains. Hybridomas generated by fusing spleen cells from IgA-primed Balb/C mice with murine myeloma (Sp2/0) were screened for IgA-specific monoclonal antibodies against a panel of ovine IgG2, IgG1, IgA and IgM. One particular clone, F3-4B4, identified as murine IgG1, was monospecific against ovine IgA with no cross reactivity against bovine immunoglobulins. This hybridoma was successfully tested as a serological probe by ELISA profiling to locate IgA containing fractions in the course of immunoglobulin purification from biological fluids.     

Isolation and characterization of the immunoglobin of pike (Esox lucius L.)

The purification of the immunoglobul in from pike serum and its physicochemical characterization is presented. The immunoglobul in was prepared by means of gel filtration and ion exchange chromatography. Measurements in the analytical ultracentrifuge showed a sedimentation constant of 15.0 S. A molecular weight of 650.000 was calculated. The immunoglobulin was composed of heavy and light chains of molecular weights 60.000 and 22.500, respectively. It is likely that the immunoglobulin of pike is composed of 8 heavy and 8 light chains and possesses a tetrameric structure. The heavy chains contain 9.2% sugars and amino sugars. The amino acid composition of the chains is similar to that of other fish immunoglobulins.     

Ovine placental eluate immunoglobulins recognise isologous and third party acid-treated trophoblast microvesicle antigens in vitro

Placental microvesicles were prepared from ovine placentae and immunoglobulins eluted with 0.5 M glycine buffer pH 2.5. The ability of eluate immunoglobulins to re-associate with isologous (self) and third party acidified microvesicles was tested by ELISA. Ovine placental immunoglobulins re-associated with isologous and third party acidified microvesicles suggesting that at least 2 types of antigenic epitopes I and II maybe expressed on the ovine placentae. Type I antigens may be present on placentae of all ovines while type II epitopes may be paternally derived, hence unique to each pregnancy. Analysis by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27 kDa, respectively, together giving a relative molecular weight of 158 kDa. Results suggest that immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the trophoblast, possibly defining a mechanism by which the foetus evades maternal immunological rejection.     

Preparation of porcine IgA and its detection using the fluorescent antibody technic]

The preparation of immunoglobulin A (IgA) from porcine colostrum, intestinal content and serum is described. The best results were achieved with colostrum, from which an antigen of satisfactory purity was prepared by purification on Sephadex G-200, on DEAE cellulose and subsequent filtration on Sephadex G-200. The serum to this antigen raised in rabbits was adsorbed to an immunoadsorbent from porcine serum (PS) or porcine IgG. The adsorbtion of the serum against secretory IgA (SIgA) to PS removed its undesirable heterologous and nonspecific reactivity. The anti-SIgA serum adsorbed in this way still reacted with IgA from porcine serum. In the direct and indirect immunofluorescent staining we detected the main antigenic determinants of the SIgA molecule, i. e. the heavy chains and the secretory component.     

顿河红豆草浓缩单宁生物合成相关蛋白鉴定

以二倍体顿河红豆草为实验材料,运用组织培养法和香草醛-盐酸法筛选优良浓缩单宁高表达的细胞系。用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)技术,对同一细胞系不含浓缩单宁(CT^-)愈伤组织和含有浓缩单宁(CT⁺)愈伤组织蛋白质进行鉴定。用制备型SDS-双向电泳对目标蛋白进行纯度分析,以及用SDS-PAGE法和IEF(等电聚焦)法测定该目标蛋白质分子量和等电点。最后经电印渍技术,将目标蛋白质转移至固相膜上进行氮端氨基酸序列分析。结果表明,筛先出10个优良浓缩单宁合成细胞系和相同基因型愈伤组织增殖细胞系。同一细胞系含有浓缩单宁愈伤组织比不含者多一条蛋白带,该蛋白分子量为28kD,等电点为5.7。该电印渍法将蛋白印渍至PVDF(聚偏二氟乙烯)膜上的效率较高。并测定了氮端部分氨基酸序列。     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.     

The metabolism and transport of bovine serum SIgA

Experiments were undertaken in six Holstein-Friesian cattle to determine whether secretory IgA (SIgA) could be transported from serum into exocrine body fluids. Preliminary data indicated that when IgG₁, IgG₂, IgM and SIgA were administered i.v., only SIgA and IgG₁ appeared in bile 90 min later at concentrations equal to or exceeding those in serum at the same time. Two hours post-injection, 70% of the SIgA recovered in bile was intact however only 30% co-precipitable with anti-secretory component (SC) while >90% of the administered IgA was precipitated by this method. All recovered IgG₁ was of low molecular weight. More detailed studies indicated that the IgA recovered in bile 7 h post-injection or in milk 3 h post-injection, was predominately lower molecular weight than intact SIgA. Most of this low molecular weight radioactivity was TCA precipitable and ca 50% was dialyzable; these data indicate that TCA-precipitability is an inadequate criterion for determining whether intact SIgA is transported. The radioactivity recovered in parotid saliva was almost entirely non-TCA precipitable and dialyzable. Almost all SIgA recovered in bovine serum remained intact and had a of 15.7 h. When transport into milk and bile was calculated from total, recovered radioactivity (i.e. 29% and 2.7, respectively), data compared favorably with those conducted in sheep in which dimeric IgA (without SC) was administered i.v. When we calculated transport on the basis of recovered intact IgA, only 1.47 and 0.54% of the injected dose had been transported into milk and bile, respectively, 24 h later. Most IgA in ruminant bile may be of serum origin although the same appears to be unlikely for the IgA in milk.