BrdU抗血清制备和应用的研究——Ⅱ.应用BrdU抗体技术显示BrdU标记的染色体和DNA

使用高度特异的BrdU抗血清,通过酶标第二抗体和DAB-H_2O_2或AEC-H_2O_2显色法,成功地显示了CHO和人染色体的BrdU标记区域。使用同样的程序和方法亦成功地显示了硝酸纤维膜上pg水平的BrdU-DNA印迹点。     

黄鳝IgM的分离纯化及兔抗黄鳝IgM抗血清的制备

目的 分离纯化黄鳝血清免疫球蛋白,制备其兔抗血清,并检测抗血清的特异性。方法 用Protein A亲和层析的方法纯化黄鳝血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价,通过western blotting检测抗血清的特异性。结果 纯化了黄鳝血清免疫球蛋白,免疫双扩散法测定兔抗黄鳝免疫球蛋白血清效价为1∶32,western blotting结果显示抗血清具有很好的特异性。结论 成功纯化了黄鳝免疫球蛋白,制备了兔抗黄鳝IgM抗血清,为建立黄鳝的血清学检测系统奠定了基础。     

人肝脏特异的F抗原分离纯化及其抗血清制备

取新鲜的人肝脏,充分洗涤,制备匀浆,上清液经Sephadex-G200、DE52层析,聚丙烯酰胺凝胶电泳,得到电泳纯Ｆ抗原,并免疫制备抗血清.     

乌鳢血清免疫球蛋白IgM的分离纯化及其兔抗血清的制备

目的 分离纯化乌鳢血清免疫球蛋白,并制备其兔抗血清。方法 用Protein A亲和层析的方法纯化乌鳢血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,测定其重链、轻链的分子量,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价。结果 纯化了乌鳢血清免疫球蛋白,SDS-PAGE测定其重链和轻链的相对分子质量分别为78×10^3和27×10^3左右,免疫双扩散法测定兔抗乌鳢免疫球蛋白抗血清效价为1∶32。结论 成功纯化了乌鳢免疫球蛋白,制备了兔抗乌鳢IgM抗血清,为研究乌鳢的免疫机制、建立乌鳢的血清学检测系统奠定了基础。     

小鼠组蛋白去乙酰化酶2多肽抗血清的制备

目的利用人工合成小鼠组蛋白去乙酰化酶2（histone deacetylase 2,HDAC2）多肽制备特异性抗HDAC2抗血清,用于相关疾病的体内外诊断。方法根据HDAC2基因编码的氨基酸序列合成多肽,与载体偶联后免疫动物,所制备的抗血清用ELISA、Western blot及免疫组织化学方法鉴定。结果 ELISA检测表明所制备的抗血清可同多肽抗原发生阳性反应,效价1∶4 000;Western blot结果显示抗血清可与多肽抗原及APP/PS1转基因小鼠的脑组织发生反应;免疫血清1∶100,1∶200,1∶400,1∶800四个稀释度均能与小鼠脑组织中的HDAC2反应。结论所制备的多肽抗血清可识别组织及血清中的HDAC2,可应用于相关领域的体内外研究。     

腮腺炎病毒抗血清的制备

以制备适用于疫苗生产检定中病毒鉴别试验和外源因子检查的高效价腮腺炎病毒抗血清为目的。用腮腺炎病毒接种SPF鸡胚尿囊腔,培养收取病毒尿液免疫SPF鸡,采集抗血清。腮腺炎病毒接种Vero细胞,培养病毒抗原经PEG沉淀,超速离心法纯化后免疫家兔采集抗血清。比较两种免疫方法所得病毒抗血清效价。结果显示SPF鸡抗腮腺炎病毒血清中和抗体GMT为1:1716,兔抗腮腺炎病毒血清中和抗体GMT为1:732。两种动物抗血清均适用于疫苗生产相关检定。免疫SPF鸡制备的病毒抗血清无特定病原及抗体污染,是毒种外源因子检测和疫苗鉴别试验的理想试剂。免疫SPF鸡制备病毒抗血清的程序简单,结果易于验证,有利于生物试剂标准化。     

蓖麻蚕卵黄磷蛋白鉴定、纯化及抗血清制备

本文通过聚丙烯酰胺凝胶电泳(SDS—PAGE)法,分析了蓖麻蚕卵黄蛋白质组分,对即黄磷蛋白(Vitellin Vn.)进行了鉴定、纯化,并制备了抗血清。对卵黄磷蛋白的分子量及相对含量进行了测定。并证明卵黄磷蛋白若处在酸性pH条件下能降解。     

大鼠精核蛋白抗血清的制备

用大鼠精核蛋白－核糖核酸复合物免疫大鼠得到了特异的抗ＲＰ抗血清,并用Ｉｍｍｕｎｏｄｏｔｔｉｎｇ和Ｉｍｍｎｕｏｂｌｏｔｔｉｎｇ方法验证了其特异性。该抗血清和ＲＰ有特异的反应,并和哺乳动物小鼠和羊的精核蛋白有一定程度交叉反应,而与体细胞类型核蛋白无交叉反应。并对制备该抗血清的意义予以讨论。     

制备抗血清方法的比较

应用抗血清同病媒昆虫胃血作沉淀反应,来查明昆虫的吸血习性及其传播疾病的重要性,在流行病学中具有重要的意义。虽然制备抗血清的方法很多,但文献中尚缺乏比较。为此,我们进行了下列试验。     

大鼠精核蛋白抗血清的制备

用大鼠精核蛋白（Ｒａｔ　Ｐｒｏｔａｍｉｎｅ,ＲＰ）－核糖核酸（ＲＮＡ）复合物（ＲＰ－ＲＮＡ　Ｃｏｍｐｌｅｘｅｓ）免疫大鼠,得到了特异的抗ＲＰ抗血清,并用Ｉｍｍｕｎｏｄｏｔｔｉｎｇ和Ｉｍｍｕｎｏｂｌｏｔｔｉｎｇ方法验证了其特异性。该抗血清和ＲＰ有特异的反应,并和哺乳动物小鼠和羊的精核蛋白有一定程度交叉反应,而与体细胞类型核蛋白（Ｈ１,Ｈ２ａ,Ｈ２ｂ,Ｈ３,Ｈ４）无交叉反应。并对制备该抗血清的意义予以讨论。     

豌豆铁蛋白的纯化及其抗血清的制备

干豌豆种子粗提物经MgCl2 盐析、AcA2 2 凝胶过滤和DEAE 纤维素阴离子交换柱层析等方法进行纯化 ,以邻菲咯啉显色法检测铁蛋白 ,最后获得纯的铁蛋白 .纯化的铁蛋白在PAGE上显示一条带 ,SDS PAGE显示该蛋白仅含 2 8kD一条亚基 .纯化的豌豆铁蛋白免疫兔 7周后 ,琼脂糖双扩散法检测抗血清效价达 1∶32 .用分级盐析法纯化抗血清 ,纯化后的抗体用琼脂糖双扩散法对大豆铁蛋白粗提物有免疫交叉反应     

盐穗木HcDMC1的原核表达和抗血清的制备

DMC1是减数分裂过程中同源染色体配对和重组修复所必需的减数分裂特异蛋白。根据盐胁迫下盐穗木转录组数据库,克隆获得的盐穗木DNA损伤修复基因命名为HcDMC1。为深入分析盐穗木HcDMC1基因的耐盐功能,通过原核表达获得盐穗木HcDMC1的融合蛋白,用于免疫小鼠制备特异性的HcDMC1抗血清。结果表明,利用pET30a-HcDMC1能够在大肠杆菌BL21（DE3）中诱导表达融合蛋白His-HcDMC1,纯化融合蛋白His-HcDMC1的含量为1.0 mg·mL^-1。每次每只小鼠免疫接种50 μg融合蛋白,三次免疫接种后进行抗体效价及特异性检测。ELISA检测抗血清滴度约为1：400 000,Western Blot检测证明了抗血清的特异性。本研究制备的小鼠抗血清能够为盐穗木HcDMC1蛋白的功能鉴定和免疫检测提供实验材料。     

Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells

Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astroglia-rich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.     

Identification as Synapsin of a Synaptosomal Protein Immunoreacting with Anti-Myelin Basic Protein Antiserum

Rat brain proteins able to react with anti-myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high-molecular-weight proteins are relatively concentrated in a synaptosome-enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin Ia and Ib. A purified synapsin preparation analyzed by immunoblot after two-dimensional gel electrophoresis also reacted with anti-myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein-conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross-reacting with extra-myelin proteins in the process of experimental allergic encephalomyelitis is considered.     

Isolation and characterization of immunoglobulin of the Indian major carp, rohu [Labeo rohita (Ham.)

Information on the structure and character of immunoglobulin of fishes is essential in health management. A study was carried out to characterize the serum immunoglobulin (IgM) of the Indian major carp, rohu Labeo rohita (Ham.). Rohu (500g) were immunised with bovine serum albumin (BSA) and the anti-BSA antibody was purified employing BSA-CL agarose affinity column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified Ig in a 3% gel under non-reduced conditions revealed a single protein having a molecular weight of 850kDa. Analysis of the purified serum in 10% SDS-PAGE under reduced conditions revealed that the immunoglobulin contained heavy and light chains with molecular weights of 85 and 23kDa, respectively. A polyclonal mouse anti-rohu IgM was prepared and used in an immunodot test which showed a specific reaction of the crude rohu anti-BSA antiserum and the purified anti-BSA IgM with BSA. Results indicate that the immunoglobulin of L. rohita is tetrameric IgM, similar to that of other fishes.     

Effects of tumour necrosis factor-alpha on BrdU incorporation in cultured human enterocytes

Bromodeoxyuridine incorporation is a useful method for studying the pattern of DNA synthesis in proliferating cells. The distribution pattern of incorporated BrdU in villus enterocytes of duodenal explants was analysed after exposure to TNFalpha in organ culture. TNFalpha caused a consistent, low level uptake of BrdU in the portion of the nucleus close to the nuclear membrane, this pattern was absent from the control cultures. As these epithelial cells are terminally arrested in G(0), the BrdU incorporation was thought not to be due to S phase DNA synthesis, but rather a response to the cytotoxic influence of TNFalpha. Microtitre plate proliferation assays of cell density and DNA synthesis were devised to study the effects of TNFalpha on confluent monolayers of the human foetal jejunal cell line I407 and the mouse fibrosarcoma cell line L929. Both cell lines showed a similar response to TNFalpha. Exposure to TNFalpha alone did not reduce cell numbers but did cause a significant increase in DNA synthesis (p < 0.05). When cycloheximtde was added in tandem with TNFalpha there was a significant reduction in cell number (p < 0.001) and level of DNA synthesis (p < 0.01) indicative of cell death. The DNA of cells exposed to TNFalpha and cycloheximide was fragmented when viewed on an electrophoresis gel. The results show that BrdU incorporation might be a good indicator of damage to the DNA of cells after cytotoxic insult. TNFalpha may be responsible for villus enterocyte damage in enteropathies such as coeliac disease and GVHR of the small bowel.     

Preparation and some properties of the haemoglobin-binding protein of bovine blood

Bovine haptoglobin was prepared from sera which showed peroxidase activity in the complex with haemoglobin. The procedure consisted of precipitation with ammonium sulphate at 0.5 saturation, affinity chromatography on Sepharose 4B coupled to bovine apoglobin, and gel filtration on Sepharose 4B. The preparation was heterogeneous, migrating in 7.5% polyacrylamide gel electrophoresis at pH 8.3 as four haemoglobin-binding bands. On immunoelectrophoresis with bovine antiserum the preparation formed a single precipitin arc with the mobility of alpha 2-globulin; the preparation was found to be a glycoprotein, the sugar moiety of which amounted to 16%.     

Catching and separating protein ligands by functional affinity electrophoresis

A new kind of affinity electrophoresis called functional affinity electrophoresis (FAEP) is a technique used to separate and/or capture proteins according to their functions in a native polyacrylamide gel. Protein A:immunoglobulin G, avidin:biotin, antibody:antigen, and concanavalin A:glycoprotein interactions are used to demonstrate this technique. Protein A, avidin, monoclonal anti-bovine serum albumin (BSA) antibody, and concanavalin A are embedded in distinct regions of a 7.5% native polyacrylamide gel. Some of each of the embedded proteins get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these proteins do not show significant electrophoretic mobility or they migrate in a direction opposite to the protein analytes, as in avidin. We clearly observe that polyclonal anti-human myoglobin antibody, biotinylated insulin, BSA, and ovalbumin (glycoprotein) are captured and separated in distinct regions of a FAEP gel by protein A, avidin, monoclonal anti-BSA antibody, and concanavalin A, respectively.     

Preparation of antiserum against a tryptic fragment (fragment A) of dynein and an immunological approach to the subunit composition of dynein.

An improved method for purifying the tryptic fragment (Fragment A) of flagellar ATPase (dynein) from sea urchin spermatozoa is described. The preparation appears homogeneous as judged by ultracentrifugation, electrophoresis on polyacrylamide gels, and immunological techniques. The molecular weight of undenatured Fragment A was determined to be 400,000 and 370,000 by the two methods of disc electrophoresis on polyacrylamide gel and sedimentation equilibrium, respectively. The fragment dissociated into two principal polypeptide chains with molecular weights of 190,000 and 135,000 when heated in the presence of sodium dodecyl sulfate. Antiserum against dynein was prepared in rabbits using purified Fragment A from the sea urchin Anthocidaris crassispina as an antigen. The specificity of this serum toward Fragment A and toward dynein was determined by double diffusion in agarose, by inhibition of ATPase activity, and by sodium dodecyl sulfate-electrophoresis of the antigen-antibody complex. This antiserum also reacted with the enzymes from two other species of sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Analysis of the precipitated antigen-antibody complex showed that the antiserum reacted specifically with the "high molecular weight" polypeptide seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude dynein fractions. This finding supports previous reports that this band derives from dynein ATPase. In our preparations, this "high molecular weight" dynein band appeared single.     

Use of pulsed-field gel electrophoresis to measure X-ray-induced double-strand breaks in DNA substituted with BrdU

The substitution of BrdU for TdR in the DNA of Chinese hamster ovary cells caused radiosensitization for both cell killing and an increase in the rate of neutral elution of the DNA. However, no radiosensitization was observed for the amount of DNA that migrated from the plug of agarose gels subjected to pulsed-field gel electrophoresis. An unexpected observation, however, was that the migration rate of BrdU-substituted DNA was relatively independent of radiation dose and was much less than that of unsubstituted DNA which migrated at a faster rate as the radiation dose increased. This difference in migration between TdR- and BrdU-labeled DNA was observed only when electrophoresis conditions were optimized for separating DNA molecules from 1 to 7 Mb. Possibly, the increase in negative charge on BrdU-labeled DNA increases the reorientation time during each pulse, with a resulting decrease in rate of migration, or radiation effects on BrdU-labeled DNA may be responsible for the decrease in migration rate.