草鱼IgM、IgD 和IgZ 的抗体制备与组织表达分析

根据已报道的草鱼免疫球蛋白IgM、IgZ 和IgD 的序列设计表达引物进行PCR 扩增, 将扩增片段克隆至表达载体pET-32a, 并在大肠杆菌Rosetta-gami (DE3)中进行诱导表达。利用亲和层析法纯化表达的重组蛋白, 然后免疫日本大耳白兔, 获得兔抗IgM、IgZ 和IgD 的抗血清。经免疫印迹检测, 表明IgM、IgZ 和IgD的表达产物能够被兔多克隆抗体特异性识别。应用兔抗草鱼IgM 和IgZ 的多克隆抗体, 对草鱼多种器官、组织提取的总蛋白进行免疫印迹检测, 在肠、头肾、中肾、皮肤、脾脏、脑、鳃和血液中都检测到IgM 和IgZ的表达。       

结核分枝杆菌肝素结合血凝素蛋白的表达、纯化及免疫学特性的初步研究

目的:克隆肝素结合血凝素(HBHA)基因,并在大肠杆菌中进行表达和纯化,利用获得的蛋白进行免疫学特性的初步研究.方法:用PCR方法从结核分枝杆菌H37Rv基因组扩增出HBHA基因片段,克隆至pMDI8-T载体中,序列测定正确后,将其亚克隆到表达载体pQE80L并在大肠杆菌DH5α中表达,表达蛋白经SDS-PAG及Western-blot分析后,亲和层析法纯化蛋白.获得的蛋白免疫BALB/c小鼠,测定血清抗体水平及IgG2a/IgG1比例.结果:克隆了HBHA基因,并成功表达该蛋白,SDS-PAGE及Western-blot分析表明表达产物正确.通过亲和层析法得到28kD纯化蛋白,与文献报道相符,诱导小鼠可使CD4+和CD8+细胞数明显增加.结论:成功获得了纯化的HBHA蛋白,明确了HBHA蛋白的免疫学特性,为进一步研究HBHA蛋白的致病机理及新型疫苗的开发提供了实验依据.     

结核分枝杆菌肝素结合血凝素蛋白的表达、纯化及免疫学特性的初步研究

目的:克隆肝素结合血凝素(HBHA)基因,并在大肠杆菌中进行表达和纯化,利用获得的蛋白进行免疫学特性的初步研究。方法:用PCR方法从结核分枝杆菌H37Rv基因组扩增出HBHA基因片段,克隆至pMD18-T载体中,序列测定正确后,将其亚克隆到表达载体pQE80L并在大肠杆菌DH5α中表达,表达蛋白经SDS-PAG及Western-blot分析后,亲和层析法纯化蛋白。获得的蛋白免疫BALB/c小鼠,测定血清抗体水平及IgG2a/IgG1比例。结果:克隆了HBHA基因,并成功表达该蛋白,SDS-PAGE及Western-blot分析表明表达产物正确。通过亲和层析法得到28kD纯化蛋白,与文献报道相符,诱导小鼠可使CD4+和CD8+细胞数明显增加。结论:成功获得了纯化的HBHA蛋白,明确了HBHA蛋白的免疫学特性,为进一步研究HBHA蛋白的致病机理及新型疫苗的开发提供了实验依据。     

兔抗麻雀IgY酶标抗体的制备

目的制备辣根过氧化物酶（HRP）标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。     

一种简便可较大量提取血清IgG的方法

用辛酸沉淀法结合应用少量阴离子交换剂纯化了四种动物抗血清及正常猪、羊血清的IgG。本法与硫酸铵法、亲和层析法纯化的IgG均呈现相同的电泳纯条带。本法简便、经济、回收率好,可较大量提取血清IgG。提纯过程保存抗体原有生物活性。     

应用A蛋白亲和层析法纯化单克隆抗体

应用Protein A亲和层析法,从采集的小鼠腹水中纯化出了抗凝血因子Ⅶ单克隆抗体,用SDS-PAGE和ELISA法分别检测了纯化后单克隆抗体的纯度及效价,结果显示,电泳为两条带,分别为免疫球蛋白G(IgG)的重链和轻链,纯化后的单克隆抗体纯度达到电泳纯,应用间接ELISA法测定腹水效价为1×10-7左右,与未纯化前无差异。结果表明,应用A蛋白亲和层析法能够得到纯度较高的单克隆抗体,适用于高纯度单克隆抗体的制备。     

鲫鱼血清和皮肤粘液IgM的分离纯化及部分性质的鉴定

采用盐析法结合葡聚糖凝胶柱 ,分离纯化鲫鱼血清IgM ;然后制备兔抗鲫鱼血清IgM多克隆抗体 ,将其偶联到Sepharose 4B上制成亲和柱 ,用于分离纯化皮肤粘液IgM。结果表明 :33%～ 4 5 %硫酸铵溶液沉淀处理可以去除鲫鱼血清中除IgM外的很多杂蛋白 ,再经葡聚糖凝胶柱纯化 ,IgM纯度可达 80 %以上 ,其重链和轻链的分子量分别为 79和 2 5kDa ;以兔抗鲫鱼血清IgM多克隆抗体亲和柱分离皮肤粘液IgM ,分离效果良好 ,IgM重链的分子量为 88kDa ;Westernblot显示兔抗鲫鱼血清IgM多克隆抗体识别的是血清和皮肤粘液IgM的重链部分。用ELISA测定鲫鱼血清中IgM含量在一年中的变化 ,结果表明IgM在春夏季的含量高于秋冬季     

抗甲肝病毒基因工程单抗分离纯化与鉴定

为克服血源免疫球蛋白制品的不足,开发了抗甲肝病毒基因工程单克隆抗体anti-HAV IgG。用无血清培养基培养rCHO工程细胞株,上清液经过rProtein A SFF亲和层析→脱盐→离子交换层析→超滤换液纯化后,所得anti-HAV IgG纯度达99%以上,比活性约100IU/mg,anti-HAV IgG活性回收率40%。所纯化的anti-HAV IgG分子量150kD,等电点8.4～9.3。免疫印迹实验证实anti-HAV IgG为人源全抗体分子。亲和层析介质rProtein A SFF确实存在亲和配基脱落问题,但通过后续纯化步骤可有效除去。在亲和层析过程中加入高盐清洗步骤,可有效降低宿主DNA残留量水平。对样品中自由巯基含量进行了测定,认为非还原电泳图谱中低分子量条带是由于抗体分子内存在自由巯基引起。用该工艺制备的anti-HAV IgG各项纯度检测指标均达到我国对基因工程产品的质量要求。     

抗草鱼免疫球蛋白单克隆抗体的研究

为了更好地了解鱼类的免疫特性,并为鱼类传染病的诊断提供标准试剂,我们进行了抗草鱼免疫球蛋白单克隆抗体的研究工作。用硫酸铵盐析法及DEAE-纤维素离子交换层析法从草鱼血清中提纯草鱼免疫球蛋白,用提纯的草鱼免疫球蛋白免疫Balb/c小鼠。经三次腹腔接种后,取免疫小鼠的     

抗生物素蛋白结合蛋白的发现及分离纯化

本文报道了在日本血吸虫感染的兔血清及日本血吸虫卵中,存在一种能与抗生物素蛋白（又称亲和素）专一性结合的蛋白质,称为抗生物素蛋白结合蛋白。该蛋白质与亲和素的结合与生物素及亲和素的糖链部分无关,并能在高ＰＨ、高浓度盐及去污剂等条件下与亲和素结合。利用ＤＥＡ－５２离子交换柱及偶联有亲和素的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱,从日本血吸虫感染的兔血清中,分离纯化了该蛋白质。提纯的亲和素结合蛋白在ＳＤＳ－ＰＡ     

病毒唑对肾综合征出血热患者特异性抗体应答的影响

在用病毒唑(Ribavirin)治疗肾综合征出血热(Hemorrhagic fever with renal syndrome,HFRS)(以安慰剂作对照)的双盲法临床试验中,用微量酶联免疫吸附技术对确诊的64例患者血清和尿液中特异性IgG及IgM抗体水平进行动态观察与分析,结果发现,病毒唑治疗组两种血清抗体水平均较安慰剂对照组低(IgG,P<0.001;IgM,P<0.05),而两组患者尿液中两种抗体水平与阳性率均无明显差异,表明病毒唑对HFRS患者抗体的生成具有抑制作作用,本文就该作用的原因与后果进行了分析评价。     

SOD猕猴桃果汁对体液免疫、血清与红细胞丙二醛水平的影响

目的 研究超氧化物歧化酶（ＳＯＤ）猕猴桃果汁姑降低血清、红细胞丙二醛（ＭＤＡ）含量及提高机体免疫球白水平的作用。方法 测定ＳＯＤ弥猴桃果汁服用前后正常妇女红细胞ＭＤＡ及血清ＭＤＡ、免疫球蛋白ＩｇＧ、ＩｇＡ、ＩｇＭ的含量。结果 ＳＯＤ弥桃果汁服用后,红细胞与血清ＭＤＡ含量显著降低血清,免疫球蛋白ＩｇＧ、ＩｇＡ、ＩｇＭ的含量。结果 ＳＯＤ猕猴桃果取用后,红细胞与血清ＭＤＡ量显著降低,免疫球蛋白ＩｇＧ、     

Presence of IgM in cutaneous mucus, but not in gut mucus of Atlantic salmon, Salmo salar. Serum IgM is rapidly degraded when added to gut mucus

In the first part of this study, cutaneous mucus of Atlantic salmon (Salmo salar) was shown to contain IgM, i.e. molecules composed of approximately 72 and 27 kDa subunits and reactive with polyclonal antisera and monoclonal antibodies made against serum IgM. Attempts to detect IgM-like molecules in gut mucus were negative, indicating the IgM is present, at best, in very small amounts. The degradation of serum IgM in mucosal secretions was examined in the second part of this study. Purified IgM from serum was rapidly digested in gut mucus at 4 degrees C. Intermediate 58, 52, 38, 35, 33 and 18 kDa breakdown fragments appeared when analysed in immunoblots. The transient fragments were further degraded to small fragments. HPLC analysis showed that only half of the added serum IgM was intact after 30 min of digestion, and after 4 h intact IgM could not be detected. Serum IgM was not degraded in cutaneous mucus, even after 17 h of incubation.     

Analysis of immunoglobulin,complements and CRP levels in serum of captive northern pig-tailed macaques (Macaca leonina)

The northern pig-tailed macaque(NPM,Macaca leonina) has become a widely used animal model in biomedical research. In this study, we measured serum immunoglobulin IgG, IgM, IgA, complement C3, C4 and CRP levels in 3-11 year old captive northern pig-tailed macaques using HITACHI 7600-20 automated chemistry analyzer in order to determine the influences of age and gender on these items. The results showed that serum IgA, IgM, C3 and C4 levels were not correlated with age(P0.05), while serum IgG levels increased progressively with age(r=0.202;P=0.045). Serum IgG, IgA, IgM and C3 levels were higher in females than in males(P0.05). Moreover, serum C3 concentration was both positively and strongly correlated with that of C4(r=0.700; P0.0001). This study provides basic serum immunoglobulin and complement data of captive northern pig-tailed macaques, which may prove useful for future breeding efforts and biomedical research.     

检测脊髓灰质炎IgM抗体捕捉法ELISA的建立及其在早期快速诊断中的应用

首次建立了用于脊髓灰质炎快速、分型诊断的血清IgM抗体捕捉法ELISA。检测了52例疑似病人,IgM阳性率为76.9％(40/52),而粪便病毒分离率仅为44.2％(23/52),前者的阳性检出率明显高于后者。做病毒分型诊断结果,与分离病毒中和试验定型的总符合率为92.86％(39/42)。检测601例来自全国各省的脊灰疑似病人血清,其阳性率波动于13％～92.3％,平均为61.1％。阳性率与收集血清的病日相关,发病0～3天收集者,阳性检出率为69.5％,4～25天者为64％,26～54天者为52％,55天以上者则未能检出。本检测方法需时1.5天,简便、敏感、特异,重复性良好,适用于脊灰的早期快速诊断。对其实用性进行了讨论。     

Serum immunoglobulins IgG, IgA and IgM in patients with oral lichen ruber

The aim of this study was to determine level of serum IgA, IgG and IgM in patients with OLR as indicators of humoral immunity which might reflect cell-mediated immunity. This study was conducted on 30 patients (age 60.17 +/- 11.75) with clinically and histopathologically confirmed diagnosis of OLR and 30 healthy controls (age 56.16 +/- 11.82) Determination of serum IgA, IgG and IgM was performed by use of standard laser nephelometry in both patients and controls. Statistical analysis was done using Mann-Whitney U test and the level of significance was determined as p values lower than 0.05. Serum IgA and IgM in patients with OLR were significantly increased in comparison to the control group, while serum IgG levels were higher in patients with OLR but they did not reach significance. We might conclude that elevated levels of serum IgA and IgM show that humoral immunity is implicated in the pathogenesis of OLR.     

Diagnostic value of dengue virus-specific IgA and IgM serum antibody detection

The diagnostic value of dengue virus (DV)-specific immunoglobulin A (IgA) serum antibody detection, by an indirect immunofluorescence assay (IFA) was evaluated. For this study, the kinetics of DV-specific IgA serum antibodies was analysed in two experimentally immunised macaques, paired samples from 35 patients suspected of a primary or secondary DV infection, paired sera from patients with high levels of IgA specific antibodies against influenza virus (n = 15), sera from patients with other viral infections (n = 40) and healthy blood donors (n = 10), which served as controls. The presence of DV-specific IgA serum antibodies in humans and in monkeys was compared with that of DV-specific IgM demonstrated in a capture enzyme-linked immunosorbent assay (ELISA). The development of DV-specific IgA and IgM antibodies in macaques proved to be similar to that observed in humans with a DV infection. In sera obtained from suspected primary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 1/6 (17%) and 6/6 (100%), whereas IgM was detected in 4/6 (67%) and 5/6 (83%), respectively. In sera from suspected secondary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 18/29 (62%) and 28/29 (97%), whereas IgM was detected in 20/29 (69%) and 28/29 (97%), respectively. The control group consisted of five paired serum samples from yellow fever vaccinated individuals and a patient with acute tick-borne encephalitis, 15 paired serum samples from patients with high levels of IgA antibodies specific for influenza virus and 40 serum samples from patients with specific IgM antibodies against other viruses. Ten serum samples from healthy blood donors were included. Among the control serum samples, in one patient, both DV-specific IgA and IgM antibodies were present, and in three sera DV-specific IgM antibodies could be demonstrated. These data suggest that detection of DV-specific IgA serum antibodies by IFA may have additional value for the diagnosis of DV infection.     

IgG autoantibody activity in normal mouse serum is controlled by IgM

In the serum of normal BALB/c mice, IgG antibody reactivity to mouse actin and tubulin, DNA, and TNP groups was very low compared to that of the IgM. This activity was considerably increased when IgG was separated, by affinity chromatography on protein A-Sepharose, whereas no difference in the IgM activity was observed. Addition of IgM to IgG isolated from the same serum resulted in the inhibition of IgG binding to these Ag. Isolation of IgG antibodies on actin, TNP, and tubulin immunoadsorbents has indicated that at least part of the IgG antibodies is polyreactive. In order to understand this inhibition better, experiments with F(ab')2 fragments of IgG were performed. IgM inhibited the binding of F(ab')2 to the antigens in a dose-dependent manner and reacted with immobilized F(ab')2. IgM isolated on F(ab')2 immunoadsorbent, as compared to the initial IgM preparation, were less active toward the Ag but more inhibitory for IgG binding to the Ag. In some pathologic situations, IgM failed to inhibit some IgG antibody activities. The anti-DNA IgG activity from (NZB x NZW)F1 mice was not affected by autologous IgM. Similarly the anti-tubulin IgG from mice infected with Trypanosoma cruzi were less inhibited by IgM from autologous serum than antitubulin IgG from normal mice. These results are compatible with the existence in normal mice of an idiotypic-like network, regulating via an IgM population in the serum, the binding of IgG autoantibodies to self Ag. Modifications of this idiotype-anti-idiotype system might lead to the expression and/or expansion of autoreactive IgG-producing clones.     

Serum immunoglobulin levels following allogeneic bone marrow transplantation

In order to study the posttransplant evolution of serum immunoglobulin levels, we measured serum IgG, IgA and IgM levels in 50 recipients of allogeneic bone marrow before transplantation and at different intervals thereafter (days 39, 120, 365 and 730). IgG and IgM levels were depressed for 1 year and IgA levels for 2 years posttransplant. Immunoglobulin deficiency was more severe and prolonged in patients with graft versus-host-disease. Hypogammaglobulinemia may contribute to the frequent infections observed in these patients, especially those with chronic graft-versus-host disease.