CSDC2蛋白免疫优势肽段分析及其多克隆抗体的制备

目的 为制备含有C2的冷休克结构域(cold shock domain containing C2, CSDC2)的特异性抗体，本试验预测了CSDC2蛋白的免疫优势肽段并制备多克隆抗体，可为进一步探究其功能奠定基础。方法 本研究以绒山羊CSDC2蛋白全长氨基酸序列为基础，应用在线分析工具Expasy分析亲疏水性；SOPMA和Predictprotein共同分析二级结构；DNAstar分析抗原指数、表面可及性、柔韧性；TMHHM 2.0预测跨膜结构；SWISS-MODEL预测三级结构；SignalP 5.0预测信号肽；NetPhos 3.1预测磷酸化位点；SVMTriP在线分析软件预测抗原表位，综上分析获得CSDC2免疫优势肽段，制备多克隆抗体；ELISA检测多克隆抗体效价，Western blot验证抗体特异性。结果 CSDC2为不稳定亲水蛋白，无跨膜结构和信号肽区域，具有22个磷酸化位点，二级结构主要由无规则卷曲、α-螺旋和延伸链构成。最终选取绒山羊CSDC2蛋白N-端15-33氨基酸序列(HSPKSPVWPTFPFHREGSR)为特异免疫肽段，诱导兔产生CSDC2蛋白特异性IgG抗...     

两性糖体液免疫应答机制的研究

分别从抗体水平和细胞水平上探寻两性糖与中性糖、糖蛋白诱发的免疫反应的差异.在抗体水平上采用间接ELISA法测定抗体效价及相对亲和常数,在细胞水平上采用MTT法测定细胞增殖情况.与中性糖相比,两性糖能明显刺激机体表达IgG类抗体,促进脾细胞增殖.两性糖表现出与糖蛋白相似的免疫应答特性,初步判断两性糖能诱使淋巴细胞的基因发生重排,出现抗体同型转换.     

5-羟色胺在F344大鼠下丘脑释放及其免疫调节作用

在F344雄性大鼠腹腔注射血蓝蛋白(KLH),研究T 细胞依赖抗原初始免疫反应对中枢神经系统5 羟色胺(5 HT)代谢的影响。当免疫刺激大鼠4天后,其下丘脑匀浆5 羟色胺水平出现降低,同时应用在体脑微透析技术研究发现清醒大鼠腹腔注射血蓝蛋白4天后,其下丘脑前叶细胞外液的5 羟色胺水平增加。为了评价在免疫反应中大鼠下丘脑5 羟色胺释放效应,本文应用氯苯丙胺(PCA)选择性预先耗竭5 羟色胺释放,并测定这些动物在注射血蓝蛋白后抗体产生情况。结果显示在应用氯苯丙胺预处理的血蓝蛋白免疫动物特异抗体IgM和IgG与未用氯苯丙胺预处理的动物比较均有明显增加(P<0.01)。上述资料表明,对于T 细胞依赖抗原,其初始抗体产生的量是由位于下丘脑5 羟色胺神经终端抑制性递质释放所调节。     

绵羊特异新基因OaN2片段的克隆及融合表达

为制备绵羊MHC区段1个新基因(OaN2)的特异性抗体及其表达模式和功能研究奠定基础,克隆OaN2的1个特殊片段(OaN2F,Genbank登录号:GQ244478)并做融合表达。以反转录得到的中国美利奴细毛羊的第一链cDNA为模板进行PCR扩增,并利用扩增产物构建重组质粒,转化感受态大肠杆菌DH5α后诱导表达得到OaN2F和GST(谷胱甘肽S转移酶)的融合蛋白GST-OaN2F,将其纯化后鉴定。结果表明,成功克隆到了OaN2F(111bp)片段,并得到了纯化的30kD的GST-OaN2F融合蛋白。     

用酶联免疫吸附试验诊断蓝氏贾第鞭毛虫病免疫价值的研究

用蓝氏贾第鞭毛虫纯培养虫株和超声粉碎可溶性虫体部分作为抗原,采用ELISA方法检测人体感染蓝氏贾第鞭毛虫血清免疫抗体并讨论其寄生虫学诊断价值．检测65份成人贾第虫病血清,其中55份IgG和／或IgA阳性,阳性率84．62％．20份10岁以下儿童贾第虫病人血清无1份阳性．结果提示蓝氏贾第鞭毛虫感染产生的体液免疫反应存在有年龄依赖性,酶联免疫吸附试验用于蓝氏贾第鞭毛虫病免疫诊断具有局限性．     

羊大片吸虫龙岩分离株ITS基因的克隆及序列分析

应用PCR方法扩增龙岩地区羊片形吸虫分离株ITS序列,经克隆、测序后获得ITS全长序列944bp,其中ITS-1为421bp,ITS-2为361bp;通过在肝片吸虫和大片吸虫ITS种间鉴别位点上的序列分析表明,从龙岩地区羊体内分离的片形吸虫虫种属大片吸虫(命名为FgLY)。与国内外大片吸虫的进化分析表明,FgLY与中国云南的2个分离株处在一个小分支,亲缘关系最近。该结果为羊大片吸虫的进一步生物学研究和片形吸虫病的预防奠定了基础。     

Cell-mediated immunity in the liver of mice vaccinated against malaria.

Mice can be protected against several species of lethal malaria infection by vaccination, and their recovery correlates well with increased anti-malarial antibody levels, particularly IgG (ref.2). However, there is also a good correlation between protection by vaccines and priming for delayed-type hypersensitivity in the skin, although there is no obvious explanation for this effect. We now report an apparent relationship between protection and a cell-mediated immune response involving the migration of various types of cell capable of killing malaria parasites in vitro to the liver. We suggest that the effect of vaccination is to bring together parasites, specific antibody and nonspecific cytotoxic cells, and that the liver may be a major site for their interaction.     

Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria

The late blood stages of the human malaria parasite, Plasmodium falciparum, carry a major surface antigen, p190, of molecular weight (Mr) 190,000. This antigenically variable protein is actively processed, first as the parasite matures and again when it is released into the blood stream and invades a new erythrocyte to initiate a cycle of growth. It elicits a strong immune response in man; all tested adult sera from endemic areas have antibodies against this protein. Our evidence indicates that purified p190 can alter the course of parasitaemia in monkeys with falciparum malaria. We have also succeeded in cloning part of the gene for p190 and expressing it in Escherichia coli. To this end we have developed a new technique, antibody select, which greatly simplifies final identification of expressing clones.     

改进双抗夹心ELISA用于筛选单抗杂交瘤细胞株

本文利用改进双抗夹心ELISA筛选一种可测定血清样品中与血浆蛋白结合的蛋白质药物的单抗。用兔抗K102多抗经亲和层析纯化并包被后,加入用10%猴血浆PBS还原后的抗原K102,再加入杂交瘤细胞株上清液和HRP-羊抗鼠IgG、底物反应液筛选阳性单抗杂交瘤细胞株。结果用改进后的双抗夹心ELISA筛成功地选出6株能稳定分泌针对K102的单抗杂交瘤细胞株,分别命名为1C9、2D7、3E2、5F4、5F6、6B9;细胞株产生的单抗用ELISA法能检测出血清样品中的K102的浓度。提示改进的双抗夹心ELISA可用于针对与血浆蛋白结合的蛋白质药物的单抗杂交瘤细胞株的筛选。     

火菇素抗体探针的制备与高灵敏度的火菇素定性技术的建立

从金针菇子实体浸提液中提取了抗癌活性物质火菇素 ,将纯化的火菇素用Freund佐剂乳化后注射到新西兰白兔体内 ,经数次加强免疫后采血并分离了抗血清 ,应用免疫学方法检测与硝酸纤维素滤膜上抗原蛋白相结合的抗体探针 ,检测极限可达 2 0 - 4 0pg的抗原信号     

An anti-idiotype vaccine against experimental schistosomiasis

Schistosomiasis is a parasitic infection of man which is widespread in tropical countries, and which so far has resisted attempts at control. We have been approaching the problem from an immunological angle. We have previously reported the production of a rat monoclonal IgG2a antibody against Schistosoma mansoni which exhibits marked cytoxicity for schistosomula in the presence of eosinophils and a high degree of protection by passive transfer in naive rats. This antibody, IPLSm1, was shown to bind specifically to a schistosomulum membrane target antigen defined as a glycoprotein of relative molecular mass 38,000 (38K), which is strongly immunogenic in schistosome infection of various animal species including man. Although theoretically the 38K protein represents an excellent candidate for a potential vaccine against schistosomiasis, the glycanic nature of the epitope recognized by IPLSm1 limits its production by DNA recombinant technology. It was, moreover, shown that, together with protective antibodies, the 38K molecule was able to induce the production of blocking IgG2c antibodies that inhibit the functional properties of IPLSm1 both in vitro and in vivo. Therefore, following Jerne's network theory, we considered an alternative approach, the possibility of immunization using anti-idiotype antibodies. In the present study, rat monoclonal anti-idiotype antibodies were produced against IPLSm1 (AB1). Anti-idiotype antibodies (AB2) were selected by their capacity to inhibit the binding of radioiodinated AB1 to its 38K target antigen. Sera from naive LOU rats immunized with a purified AB2 preparation contained specific anti-schistosome antibodies (AB3) which bound to 38K. AB3 antibodies were strongly cytotoxic for schistosomula in the presence of rat eosinophils and conferred highly significant protection by passive transfer. Most importantly, rats immunized with AB2 demonstrated marked protection (50-80%) to a challenge infection.