东方田鼠抗日本血吸虫抗体水平检测与分析

东方田鼠具有抗日本血吸虫病的特性，观察东方田鼠感染日本血吸虫尾蚴后的特异性抗体水平变化，可为阐明其抗病机制提供依据。本试验用间接ELISA方法检测东方田鼠抗成虫可溶性抗原（SWAP）和几种日本血吸虫基因重组抗原（Sj28-GST、LHD-SjTP1、C-SjParamyosion）的特异性抗体水平动态变化，并检测东方田鼠感染日本血吸虫尾蚴前、后的IgG3亚类特异性抗体水平。结果东方田鼠在攻击日本血吸虫尾蚴后，抗SWAP的特异性抗体水平有所升高，而抗几体基因重组抗原的特异性抗体水平低，没有显著变化。东方田鼠正常血清中抗SWAP和IgG3亚类抗体的OD值是牛血清白蛋白（BSA）对照的31倍。结果表明，在东方田鼠血清中有较高水平的SWAP的IgG3亚类抗体，值得进一步深入探讨。     

感染日本血吸虫前后东方田鼠血清补体的动态变化和杀虫作用

东方田鼠是迄今为止发现的唯一一种具有天然抗日本血吸虫病特性的哺乳动物,但具体抗病机制尚不明确。本研究应用组织培养法比较了东方田鼠和BALB/c鼠感染日本血吸虫尾蚴后1、3、8、12、15 d的皮肤、肺脏和肝脏中童虫回收情况,结果表明东方田鼠对日本血吸虫的杀伤主要发生在感染后1～3 d和8～12 d。应用RT-PCR和ELISA检测东方田鼠和BALB/c鼠的补体相关分子C3、C9、CD59和DAF的转录水平,结果显示BALB/c小鼠各指标变化不明显,东方田鼠感染后8 d各因子转录水平显著升高。体外杀虫试验结果显示,东方田鼠正常血清和灭活补体血清孵育的日本血吸虫童虫死亡率分别为85.50%±7.15%和60.99%±8.61%,差异具有显著统计学意义(P 0.001)。结果表明东方田鼠补体对日本血吸虫童虫具有显著杀伤效果,东方田鼠早期对日本血吸虫的杀伤作用可能和补体相关。     

日本血吸虫基因重组抗原rSj06868对小鼠的免疫保护效果

为克隆、表达日本血吸虫假想蛋白SJCHGC068068编码基因（暂命名为Sj06868）并观察重组抗原的免疫预防效果。应用PCR技术从日本血吸虫7 d童虫、14 d童虫mRNA反转录制备的cDNA中克隆到Sj06868基因的46-438 bp DNA片段,BLASTn分析未发现其他物种中的同源性序列,也未发现其保守序列和相关功能结构域。以pET32a为表达载体、Sj06868基因在大肠杆菌Rosetta（DE3）中获得高效表达,表达产物rSj068068分子量为36 kDa,能被感染血吸虫14 d兔血清识别。将纯化的重组抗原与佐剂ESSAI ISA 206 CELL混合后免疫BALB/c小鼠,与佐剂对照组和PBS对照组相比,分别获得了31.63%、29.19%减虫率以及55.63%、56.27%肝脏虫卵减少率。用ELISA检测各组血清中抗rSj068068特异性抗体结果显示,免疫组在攻击感染前、佐剂对照组和PBS对照组在感染后均产生了较高水平的特异性抗体。本研究结果说明,Sj068068是日本血吸虫特有的童虫期高表达基因,rSj068068在抗血吸虫疫苗和血吸虫感染的诊断方面均有潜在应用价值。     

东方田鼠和小鼠感染日本血吸虫后组织与血清蛋白组分差异比较研究

探讨东方田鼠和小鼠感染日本血吸虫前后不同时点组织与血清中蛋白质电泳谱的变化。分别提取东方田鼠感染日本血吸虫(0、7、12、25 d) 和小鼠感染日本血吸虫(0、7、12、45 d)后的肝脏、脾脏、肺脏蛋白及血清进行聚丙烯酰胺凝胶电泳,对结果比较分析并进行组织病理形态学观察。结果显示,小鼠感染日本血吸虫第7和12天后肝脏150 ku附近蛋白质条带较东方田鼠表达量明显增高;东方田鼠在感染日本血吸虫后40~50 ku附近蛋白质条带较正常东方田鼠表达明显上调;感染第7天后肺脏40 ku附近蛋白质条带较其他时间点变化明显;小鼠感染日本血吸虫第7、12和45天后血清中IgG类蛋白质表达量较东方田鼠显著增高。东方田鼠和小鼠在日本血吸虫感染后不同时点间的组织及血清蛋白组分存在差异,东方田鼠肝脏和血清中150 ku附近蛋白条带较其他动物特异,蛋白质表达丰度高,该蛋白可能与东方田鼠抗日本血吸虫相关。     

东方田鼠肝脏T7噬菌体展示cDNA文库的构建

本项研究的目的是构建东方田鼠肝脏T7噬菌体展示cDNA文库,为筛选东方田鼠抗血吸虫病抗性相关基因奠定基础.用TRIzol试剂提取东方田鼠肝脏总RNA,分离纯化mRNA,经反转录合成双链cDNA.在双链cDNA末端加上EcoRⅠ/HindⅢ定向接头并用EcoRⅠ和Hind Ⅲ酶切,使其两端分别带EcoRⅠ和HindⅢ粘性末端.用Mini Column纯化、收集300 bp以上的双链cDNA片段,再连接于带有EcoRⅠ和HindⅢ末端的T7 Select 10-3b载体,经体外包装后,以BLT5403为受体菌构建T7噬菌体展示cDNA文库.经测定,库容量为1.3×107 PFU/mL,扩增后文库滴度为1.8×1011 PFU/mL.对从原始文库中随机挑取的100个噬菌斑进行PCR鉴定,重组率为91.7%,阳性克隆片段大小分布在200 bp～1 000 bp,其中有95.5%的插入片段大于300 bp.用日本血吸虫童虫可溶性抗原对文库进行了初筛,得到了21个ESTs,将这些阳性噬菌体克隆和血吸虫童虫共培养,其中大部分克隆诱导的童虫死亡率比阴性噬菌体对照高出2%～13%.     

三种抗原对家畜日本血吸虫病诊断价值的比较

以日本血吸虫基因重组抗原LHD-Sj23／pGEX、可溶性虫卵抗原（soluble egg antigen，SEA）及SEA经Sephadex G-200柱层析分离后得到的第一峰SEAl作为诊断抗原，应用ELISA检测人工感染日本血吸虫绵羊血清及自然感染日本血吸虫水牛血清。结果显示，三种抗原用于检测人工感染日本血吸虫绵羊血清的阳性符合率分剐为88．79％、98．15％、100％，阴性符合率均为100％；自然感染日本血吸虫水牛血清的阳性符合率分别为80％、80％、84％，阴性符合率分别为88．9％、93．3％、91．1％；三种抗原与伊氏锥虫病牛血清无交叉反应；检测感染日本血吸虫不同时间绵羊血清，发现感染6周后均被检出针对这三种抗原的特异性抗体，且SEA及SEAl在感染4周后即可检出特异性抗体。三种抗原的诊断效果差异不显著。     

三种试验检测东方田鼠抗日本血吸虫抗体的分析

应用薄膜尾蚴抗原片的免疫酶染色试验(IEST)、可溶性尾蚴抗原的ELISA(SCA-ELISA)和可溶性成虫抗原的ELISA(SAA-ELISA)对人工繁育东方田鼠的抗日本血吸虫抗体进行检测.结果显示阳性检出率高低依次为SCA-ELISA(71.6%,48/67)、IEST(71.4%,50/70)和SAA-ELISA(68.7%,46/67).三种试验的阳性率无显著性差异(x2=0.180,P＞0.05).IEST与SCA-ELISA、IEST与SAA-ELISA及SCA-ELISA与SAA-ELISA的总符合率分别为91.2%(62/68)、86.6%(58/67)和89.2%(58/65).卡方配对检验结果表明,三种试验中任何两种方法的检查结果相比一致.     

东方田鼠感染日本血吸虫前后血常规和血清生化指标的动态变化

东方田鼠具有天然抗日本血吸虫病特征,为了探索东方田鼠抗日本血吸虫机制,本研究分析了东方田鼠和小鼠感染日本血吸虫尾蚴前后血液细胞以及生化指标的动态变化。东方田鼠和BALB/c鼠定量感染日本血吸虫尾蚴,于感染后第0、1、3、5、8、12、16、21、28、35、42 d分别采集抗凝血和血清,进行血常规和血清生化分析。血常规检测结果表明,东方田鼠感染后第3~16 d,WBC数量显著升高(Neu数量显著升高),而小鼠血常规检测结果正常;血清生化结果显示,东方田鼠感染日本血吸虫后ALB、HDL-C显著升高,小鼠各指标变化不明显。本研究结果显示东方田鼠天然免疫细胞Neu和血清ALB可能与其抗日本血吸虫特性相关,对补体杀伤日本血吸虫机制研究具有重要的指导意义。     

日本血吸虫7d童虫cDNA文库构建及免疫筛选

为了从7 d童虫cDNA文库筛选出童虫发育阶段特异性表达抗原基因。本研究首先构建了日本血吸虫7 d童虫cDNA文库,其次再采用日本吸血虫感染血清进行筛选。结果显示:建立的文库插入片段为0.5~3.0 kb,文库重组率为98%,初始文库滴度为2.4×10~6 pfu/mL,扩增后滴度为1.6×10~9 pfu/mL,利用感染日本血吸虫10 d和42 d的小鼠血清免疫筛选该文库,初筛和复筛后获得17条有效EST序列,其编码7个基因,分别为复制蛋白(2 ESTs)、肝再生增强因子(3 ESTs)、血小板活化因子(6 ESTs)、钙调理蛋白基因(3 EST)、丝束蛋白(1 ESTs)、核糖体RNA(1 EST)和还原型辅酶脱氢酶基因(1 EST)。该研究结果对今后探讨血吸虫的生长发育机制及筛选血吸虫早期诊断抗原具有重要意义。     

感染日本血吸虫自愈水牛血清抗日本血吸虫的研究

为了明确自愈水牛血清中是否存在抗日本血吸虫的天然分子,为水牛感染日本血吸虫后自愈机制的阐明提供新的线索,试验在日本血吸虫童虫培养基中加入自愈水牛血清,使其终浓度为50%,每个样本设3个平行孔,以含兔血清的DMEM培养基为空白对照,含健康水牛血清的DMEM培养基为阴性对照,96 h内连续观察各组培养基中的童虫活性,镜检统计童虫死亡率,观察自愈水牛血清对日本血吸虫的杀伤活性。结果表明:自愈水牛血清体外具有明显的杀伤日本血吸虫童虫活性,其杀虫率分别为(23.0±4.0)%、(25.6±1.6)%、(20.6±1.4)%,与健康水牛对照比较差异显著(P0.05)。说明自愈水牛血清具有体外杀伤日本血吸虫童虫活性。     

孔雀IgG与鸡IgG的初步比较研究

通过比较孔雀 Ig G与鸡 Ig G的相似性 ,证实鸡疾病诊断试剂用于孔雀疾病诊断的可行性。用饱和硫酸铵盐析法提纯鸡 Ig G、孔雀 Ig G,比较 Ig G粗提物的 SDS-聚丙烯酰胺凝胶电泳图谱。用直接 EL ISA方法分析孔雀 Ig G、鸡 Ig G与抗鸡 Ig G结合程度的差异 ,并用蛋白免疫转印法进一步验证二者与抗鸡 Ig G的结合情况。最后 ,应用数种鸡疾病诊断试剂盒及血凝抑制实验进行孔雀疾病的血清学调查及诊断应用初步研究。结果表明 :孔雀 Ig G与鸡 Ig G具有较大相似性 ,并能够与兔抗鸡酶标二抗结合 ,但结合力弱于鸡 Ig G;可应用鸡疾病诊断试剂盒诊断孔雀疾病     

Production and characterization of monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2

Monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2 were produced using conventional cell fusion techniques. Monoclonal antibodies detected by preliminary screening assays were further characterized and their specificity verified by titration of ascites in radioimmunoassay or passive haemagglutination using pure sheep IgG1 or IgG2. Further evidence for the subclass specificity of the antibodies was obtained from immunoelectrophoretograms of sheep serum or purified proteins developed with monoclonal antibodies. Reaction of monoclonal antibodies with various IgG fragments showed that the determinants recognised were located on the pFc' portion of the heavy chain.     

Separation of ovine IgG1 and IgG2 on protein A-sepharose

A method using protein A-Sepharose chromatography was developed to separate and purify ovine IgG1 and IgG2. The IgG1 eluted from protein A-Sepharose at pH 6.8 and IgG2 eluted at pH 4.5. This method was used to show the specific transfer of IgG1 from the colostrum to newborn lambs. After separation on protein A-Sepharose both IgG1 and IgG2 were pure as analyzed by isoelectric focusing, Western Blotting and SDS-PAGE. The isoelectric points for the immunoglobulins were calculated to be 3.5 for IgG2 and a range from 6.2 to 8.1 for IgG1. The subclass, IgG1, was present in the whey and was the subclass that was found in the serum of lambs after being fed colostrum. The ewe sera had a decrease of both IgG1 and IgG2 at the time of lambing compared to 2 weeks prior to parturition.     

Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG1 and IgG2.

Ovine bone marrow-derived macrophages (BMM) may express several IgG receptor (Fc gamma receptor; FcR) subsets. To study this, model particles (opsonized erythrocytes; EA), which are selectively handled by certain FcR subsets of human macrophages were used in cross-inhibition studies and found to react in a similar manner with FcR subsets of sheep macrophages. In experiments with monoclonal antibodies against subsets of human FcR, human erythrocytes (E) treated with human anti-D-IgG (anti-D-EAhu) and sheep E treated with bovine IgG1 (Bo1-EAs) were handled selectively by human macrophage FcRI and FcRII, respectively. Rabbit-IgG-coated sheep E (Rb-EAs) were recognized by FcRI, FcRII and possibly also by FcRIII of human macrophages. Anti-D-EAhu, Bo1-EAs and Rb-EAs were also ingested by sheep BMM. Competitive inhibition tests, using various homologous and heterologous IgG isotypes as fluid phase inhibitors and the particles used as FcR-specific tools in man (anti-D-EAhu and Bo1-EAs), revealed a heterogeneity of FcR also in sheep BMM. Thus, ingestion of anti-D-EAhu by ovine BMM was inhibited by low concentrations of competitor IgG from rabbit or man in the fluid phase, but not at all by bovine IgG1, whereas ingestion of Bo1-EAs was inhibited by bovine IgG1. This suggested that anti-D-EAhu were recognized by a FcR subset distinct from that recognizing bovine-IgG1. It was concluded that sheep BMM express functional analogs of human macrophage FcRI and FcRII and that Bo1-EAs and anti-D-EAhu are handled by distinct subsets of BMM FcR. All EAhu tested (EAhu treated with anti-D, sheep IgG1 or sheep IgG2) were ingested to a lower degree than EAs. This inefficient phagocytosis could be enhanced by treatment of EAhu with antiglobulin from the rabbit, suggesting that it is caused by a low degree of activity of opsonizing antibodies rather than special properties of the erythrocytes themselves. Several lines of evidence suggested that both FcR subsets of ovine BMM recognize both ovine IgG1 and IgG2. In contrast, bovine IgG1 reacts with one FcR subset and bovine IgG2 interacts inefficiently with all FcR of ovine BMM.     

Serum levels of the immunoglobulins IgG and IgG(T) in horses

Levels of the immunoglobulins IgG and IgG(T) in serum in Norwegian horses of the breeds “Døle” and “Fjord” were determined by the quantitative radial immunodiffusion test.No significant differences were apparent between the 2 Norwegian breeds. The immunoglobulin levels were approximately in the same range as previously reported for Shetland ponies.Immunoglobulins could not be detected in the newborn foal. As early as 24 hrs. after birth the mean immunoglobulin level was within the adult range. After a drop during the first month of life, the immunoglobulins increased. IgG(T) rose more rapidly and to a higher level than IgG.In 2 year old horses, IgG(T) was significantly higher than in adults, while IgG was significantly lower. IgG(T) seems to be a very important immunoglobulin in foals and young horses.     

牛初乳免疫球蛋白对仔猪血清IgG含量和增重的影响

试验选用120头大白仔猪,随机分为3组。A组为对照组,饲喂生理盐水,其余各组均于出生后24 h之内饲喂不同剂量的牛初乳免疫球蛋白制剂,观察对仔猪5、10日龄血清中牛IgG和猪IgG含量以及断奶时增重的影响。结果表明,饲喂牛初乳免疫球蛋白制剂4 g的仔猪,其5、10日龄血清中牛IgG和猪IgG含量显著高于对照组(P<0.05),断奶时增重显著高于对照组(P<0.05)。     

牛初乳中免疫球蛋白IgG的检测

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Immunohistochemical localization of IgG1 and IgG2 in prepartum and lactating bovine mammary tissue

The differential distributions of IgG1 and IgG2 were determined in prepartum and lactating bovine mammary tissue by indirect immunofluorescence. IgG1 was found predominately within the alveolar epithelial cells and lumens of prepartum tissue whereas IgG2 was largely confined to the stromal area surrounding the alveoli. Both IgG subclasses were confined predominately to the stroma in lactating tissue. Few IgG containing stromal cells were readily distinguished in any of the mammary tissue used in this study.     

Idiotype expression of IgG1 and IgG2 in dogs naturally infected with Leishmania infantum

Here we analyzed, by Western blot analysis, the idiotype expression of IgG1 and IgG2 in 109 canine sera corresponding to 50 dogs from endemic areas of leishmaniosis in order to detect markers related to Leishmania infantum infection and clinical condition (asymptomatic or symptomatic). Twenty-four dogs from an area free of leishmaniosis were used as controls. IgG1 and IgG2 responses in symptomatic and asymptomatic L. infantum infections differed mainly in subclass production (ELISA values), with higher IgG2 production occurring particularly in symptomatic dogs. Nevertheless, we observed little difference in the idiotype expression of these IgG subclasses, which, in general, recognized the same antigenic fractions. While early L. infantum infection was characterized by recognition of polypeptide fractions of low molecular weight, mainly fractions of 14, 16 and 18 kDa by IgG1 and 14 and 16 kDa by IgG2, symptomatology was associated with recognition by both IgG subclasses of a 24 kDa fraction and other antigens belonging to the AG24 family.