人血型糖蛋白A衍生物对恶性疟原虫裂殖子识别及入侵红细胞的效应

用电泳纯人血型糖蛋白A(GPA)制得下列衍生物:(1)用胰蛋白酶酶解GPA分离得GPA糖肽;(2)制备GPA和GPA糖肽两种抗体;(3)制备去糖GPA(dGPA);(4)用人红细胞膜全脂分别重组成含GPA及dGPA的两种脂质体。用上述制品对FCC-1/HN株恶性疟原虫裂殖子实验,发现:(1)GPA脂质体可与裂殖子结合,而dGPA脂质体呈阴性反应。(2)GPA,GPA糖肽,GPA抗体,GPA糖肽抗体及GPA脂质体均有阻止恶性疟原虫裂殖子入侵人红细胞的效应。     

恶性疟原虫单克隆抗体保护性机制的实验研究

用体外抑制试验、调理试验和细胞毒试验等方法,对抗恶性疟原虫红内期McAb之功能特性进行了研究。17株McAb中有7株对同步化培养的恶性疟原虫的体外增殖具有明显的抑制作用;抑制强度取决于剂量和作用时间。在McAb—IgG浓度为0.6mg/ml时,上述7株McAb的抑制活性均在94％以上。在6株McAb中,有4株能促进巨噬细胞吞噬疟原虫而发挥调理作用。在7株McAb中,有4株能加强腹腔细胞杀伤和杀死疟原虫而表现出细胞毒活性。结果提示,恶性疟原虫McAb似有“多功能”、“双功能”和”单功能”之分,用“多功能性McAb”纯化的疟疾抗原,在制备疟疾亚单位疫苗方面可能更有意义。     

红细胞血型糖蛋白A位点正常变异及肿瘤放疗的影响

目的了解血型糖蛋白Ａ（ＧＰＡ）背景突变水平,观察照射近期的变化规律。方法在正常人和肿瘤放疗病人中进行ＧＰＡ突变分析,结合单克隆抗体标记和流式细胞技术,检测ＭＮ杂合子个体中罕见的变异细胞ＮＯ和ＮＮ,确定变异率。结果对２８个正常成人的初步测定,ＮＯ／ＮＮ变异率（Ｖａｒｉａｎｔｆｒｅｑｕｅｎｃｙ,Ｖｆ）（８．３±４．１３）／（５．５０±３．７７）×１０－６;ＮＮ变异率与年龄相关性接近显著意义（Ｐ＝０．０５７）,男女之间、吸烟与不吸烟者均无显著差异。随访肿瘤病人,６例治疗前ＧＰＡ变异率ＮＯ（１２．９±６．６０）×１０－６,ＮＮ（７．７±５．０１）×１０－６,与同年龄者无显著差异。连续盆腔照射１月Ｖｆ已有升高,照射２个月的病人ＮＮＶｆ（１７．５±８．９５）×１０－６,显著高于未照病人（Ｐ＝０．０４６８）。结论正常人ＧＰＡ变异率基本与文献相符,年龄等因素可影响背景水平;病人照射后Ｖｆ变化不一,表明个体对辐射敏感性不同,照射近期主要是ＮＮ的变化,是较晚造血周期细胞突变的表现。     

Mechanisms of Protective Activities of Monoclonal Antibodies Against Malaria Parasite Plasmodium Falciparum

A range of monoclonal antibodies specific for Plasmodium falciparumwere tested in vitro for their abilities to inhibit the multiplication of a partiallysynchronized culture of P.falciparum,to augment the phagocytosis of theparasites by macrophages,and to enhance the hilling of parasites by peritonealcells depleted of adherent cells.Seven of 17 monoclonal antibodies,ranging fromculture supernatant and ascitic fluid to purified IgG,were shown to have a dose-and time-dependent inhibition of the parasite growth in vitro.At a concentrationof 0.6mg/ml,the inhibitory capacity of these monoclonal IgG was above 94% overa 3-day culture period,much higher than that of the relevant polycloaal IgG.Four of six monoclonal antibodies tested augmented the phagocytosis of theparasite by macrophages,resulted from the opsonization of the parasites.Four ofthe seven monoclanal antibodies examined showed cytotoxic activity on malariaparasites.Peritoneal cells depleted of adherent cells were capable of killing theparasites in the presence of monoclonal antibodies.The results indicate that theremay be“monofunction”,“bifunction”,and“multifunction”types of monoclonalantibodies against P.falciparum.The putative protective antigen of malariaparasite purified by“multifunctional monoclonal antibody”affinity chromatographymay have potential interest as a vaccine against the parasite.     

血型糖蛋白A在恶性疟原虫入侵红细胞中的受体作用

人红细胞膜血型糖蛋白A(GPA)与恶性疟原虫入侵红细胞关系密切。本实验在体外培养条件下,观察了GPA及其抗体,α1-酸性糖蛋白、鸡卵类粘蛋白及麦胚凝集素等对恶性疟原虫入侵红细胞的影响。结果表明:GPA与恶性疟原虫裂殖子之间的结合具有高度特异性和亲和力,有饱和趋势,结合后产生特定的生物效应。首次从配体—受体相互作用的特点,证实了GPA是恶性疟原虫裂殖子识别和结合的受体。     

恶性疟原虫EBA-175与血型糖蛋白A结合位点的鉴定

目的 探索EBA-175与GPA之间的结合信息,为疟疾短肽疫苗及拮抗药物的研制奠定基础.方法 以EBA-175重组蛋白为靶,采用亲和筛选法对噬菌体随机十二肽库进行3轮筛选,通过ELISA、竞争抑制试验、Dot-ELISA及Western blotting等方法鉴定获得的噬菌体短肽与EBA-175之间的结合特性.对阳性克隆进行DNA序列测定,推导其十二肽的氨基酸序列并与GPA氨基酸全序列进行了同源性比较.结果 经3轮亲和筛选后,结合噬菌体得到良好富集.从第3轮洗脱液铺制的琼脂板中随机挑取30个噬菌体单克隆,ELISA检测有27个为阳性,阳性率达90%.竞争性ELISA显示多数阳性噬菌体能竞争抑制EBA-175与其单抗结合.DNA及氨基酸序列分析表明24个噬菌体展示十二肽中共有序列IRR与GPA的114-116位氨基酸同源.结论 阳性噬菌体表达的短肽是EBA-175所识别的模拟表位,IRR几位氨基酸可能对EBA-175与GPA的结合起重要作用.     

5株抗恶性疟McAb的免疫荧光特性及其靶抗原鉴定

本文观察了5株抗恶性疟McAb的免疫荧光特性,并对其抗原进行了鉴定。结果表明5株McAb主要与恶性疟原虫红内期发育晚期虫体的表面膜抗原发生免疫反应,免疫荧光图象为明亮的点状、均匀片状和蜂窝状。它们在对靶抗原的识别上与免疫荧光的特点呈现一致性,主要能识别恶性疟原虫红内期发育晚期虫体的~(125)Ⅰ-表面标记抗原,其中McAbs 93A_3、94B_5、92D_4和93D_4主要识别分子量为125、115、83、74和67KDa的表面抗原,而McAb94D_1则主要识别分子量为200和19KDa的抗原。     

恶性疟原虫体外培养的同步化方法

本文就恶性疟原虫体外培养的同步化方法加以综述,主要对恶性疟原虫的同步化方法,如传统的浓度分离法、渗透压分离法、Percoll梯度法、温度周期法、阿非迪霉素法,及近几年发展起来的磁性分离法及流式细胞分析仪法进行了介绍.     

Effects of derivatives of human glycophorin A and their antibodies on the recognition and invasion of Plasmodium falciparum into erythrocytes

Electrophoresis-purified human glycophorin A(GPA)was used to produce its derivatives:(1)separation and purification of glycopeptides from GPA were performed after trypsin-digestion:(2)prepanation of GPA antibody and GPA glycopeptide antibody;(3)preparation of deglycosylatedGPA(dGPA);(4)incorporating GPA or dGPA into human RBC membrane lipids to form twokinds of liposomes.The products described above were used to test Plasmodim falciparumFCC-1/HN merozoites for their ability to invade human erythrocytes.It was found that GPA-liposomes were able to bind with merozoites and dGPA-liposomes had a negative reaction.GPA,GPA glycopeptide,GPA antibody,GPA glycopeptide antibody and GPA-liposome all had the effectto hinder the invasion of merozoites into human erythrocyte,whereas dGPA-liposome had no suchan effect.     

恶性疟原虫不同虫株间环子孢子蛋白基因多态性分析

本文采用ＰＣＲ方法扩增了恶性疟原虫云南株（ＰＦＤ－３／ＹＮ）环子孢子蛋白（ＣＳＰ）部分基因，将扩增的基因片断克隆于Ｍ１３测序载体进行了基因序列测定。用ＰＣＧＥＮＥ软件对恶性疟原虫不同虫株ＣＳＰ基因序列进行了一系列比较分析。其结果表明恶性疟原虫虫株间ＣＳＰ基因存在多态性，ＰＦＤ－３／ＹＮ株ＣＳＰ基因序列与Ｔ４、Ｗｅｌｃｏｍｅ、ＮＦ５４、３Ｄ７及７Ｇ８等虫株均有不同程度的差异。     

体外连续培养FCC-1/HN株恶性疟原虫红内期超微结构改变的透射电镜观察

将1979年正式定名建株到现在,其间经历了9年反复冻存、复苏、培养的FCC-1/HN株恶性疟原虫,用培养效果明显优于成人血清的10%脐带血清进行体外培养。60天后取培养物用透射电镜观察了疟原虫和其宿主细胞——红细胞近期的超微结构情况。观察结果表明与以往已报道的同株疟原虫相比,除见到了仍有相似之处外;还发现有某些不同的特征性改变:①疟原虫寄生的红细胞,其表面结节完全消失;也未见到典型的茂氏裂隙。②疟原虫虫体胞质内空泡结构和胞质裂隙增多。提示:疟原虫和被寄生的红细胞的超微结构在外界环境条件改变时,也会随之发生一些变化。这些变化,应引起我们今后在以体外培养研究疟原虫的生物学、生理生化和药物筛选中的重视,尤其是免疫学的重视。     

红细胞感染恶性疟原虫后游离钙浓度变化的研究

采用活细胞钙离子荧光指示剂Fluo-3负载感染恶性疟原虫（FecI株）红细胞及正常红细胞，经流式细胞仪测定，根据荧光强度不同表示红细胞内游离钙浓度的变化。结果表明，感染红细胞内游离钙浓度分别为正常红细胞的1．83和2.99倍，呈明显增高。     

恶性疟原虫红内期体外同步化培养实验观察

本文定时观察了用秋水仙碱处理体外培养恶性疟原虫后的红细胞感染率及环状体、大滋养体和裂殖体各期的比例,并与用山梨醇处理进行了比较。结果发现,秋水仙碱处理后疟原虫可维持2～3个生物周期的同步化发育,并且处理后的红细胞感染率可在14％以上。山梨醇处理后疟原虫可维持2个生物周期的同步化发育,最高红细胞感染率为7.7％。     

恶性疟原虫FCCl／HN株LDH基因的克隆及序列分析

目的 克隆并测定恶性疟原虫海南(FCCl／HN)株LDN基因序列,比较FCCl/HN株与国外分离株LDH基因序列的差异。方法 根据LDH基因已知序列设计合成一对引物,应用PCR技术从FCCl/HN株基因组DNA中扩增LDH基因,并将其克隆入pMDl8-T载体。用双脱氧链末端终止法进行基因序列测定,并用分子生物学软件进行不同分离株LDH基因序列的同源性比较。结果 PCR扩增得到特异的FCCl／HN株LDH基因片段并构建了pT—LDH重组质粒。恶性疟原虫FCCl／HN株LDH基因全长951bp,编码316个氨基酸。序列分析表明,我国恶性疟原虫FCCl／HN株与国外的3所、Honduras 1株LDH基因编码氨基酸序列完全一致。结论 成功克隆恶性疟原虫FCCl,HN株LDH基因。序列测定及同源性分析表明,FCCl/HN株与其它分离株的LDH基因序列高度保守。     

应用(Dig)-DNA探针进行恶性疟流行病学调查

重组的恶性疟原虫DNA片段用洋地黄毒苷配基标记后作为探针(pPF_(14)-F-Dig),以斑点杂交试验对海南省恶性疟流行区人群的274份血样进行检测,并同时作厚薄血片镜检。结果显示,274例样本中血片镜检阳性1例,带虫率为0.36%;pPF_(14)-F-Dig 探针检出阳性15例(其中1例为镜检阳性者),阳性率为5.47%;pPF_(14)-F-Dig 与镜检的阳性符合率为1/1,阴性符合率为94.87%。每张硝酸纤维素膜可查96份样本,故该探针具有用于大规模流行病学调查的价值。     

Preparation and Identification of Rat Monoclonal Anti-idiotope Antibody to the Antibody against Erythrocytic Stages of Plasmodium Falciparum

A hybridoma cell line secreting monoclonal antibody to idiotope of the monoclonalantibody 94D1 specific for asexual erythrocytic stages of Plasmodium falciparum was established byfusion of SP2/0 mouse myeloma cells with spleen cells of Wistar rats immunized with monoclonalIgG of 94D1 purified from ascitic fluid of BALB/C mouse by affinity chromatography on ProteinA- Sepharose CL- 4B. Specificity of the anti - idiotope antibody (anti - Id), 41RF5, was deter-mined with indirect enzyme-linked immunosorbent assay (ELISA). The results showedthat 41RF5 reacted only with 94D1 IgG2b, not with normal mouse IgG and other seven mousemonoclonal antibodies - five specific for erythrocytic stages of P. falciparum, one for erythrocyticstages of P. inui, and one for Dengue fever virus type III, which indicates that 41RF5 does recognizespecifically the idiotope of 94D1 monoclonal antibody. In 41RF5 heterohybrid culture supernatant,anti-Id titre measured by ELISA was above 1:1 280. Up to the present, the heterohybrid cell linehas been cultured stably for 16 months.     

Sequence determination of exp- 1 Gene of Plasmodium Falciparum Isolate FCC1／HN

目的 测定恶性疟原虫FCCl／HN株exp-1基因序列。方法 根据exp-1基因已知序列设计合成1对引物，用PCR技术从FCCl／HN株基因组DNA中扩增exp-1基因；将exp-1基因克隆入pMD-18T载体，转化大肠杆菌JMl09感受态细胞，铺x—gal LB平板；挑取阳性菌落，用酶切，PCR扩增进行鉴定。以正确的重组质粒为模板，用双脱氧链末端终止法测定exp-1基因序列。结果 从恶性疟原虫FCCl／HN株基因组DNA中获取exp-1基因，成功克隆入pMD-18T载体；测序表明FCCl／HN株exp-1基因全长937bp，编码162个氨基酸。结论 克隆了恶性疟原虫FCCl／HN株exp-1基因，并测定了其核苷酸序列，为进-步研究其功能奠定基础。     

Antigenic variation and cytoadherence of PfEMP1 of Plasmodium falciparum-infected erythrocyte from malaria patients

Objective To approve a theoretical basis for the molecular pathogenesis of human cerebral malaria and treatment with prevention.Methods The blood samples were collected from 24 patients with cerebral malaria, 143 wit h falciparum malaria, 34 with vivax malaria and 20 healthy controls from the end emic areas of Yunnan Province, China. Using the sodium dodecyl sulfate-polya crylamide gel electrophoresis (SDS-PAGE) technique, we determined the molecular mass (Mr) of these Plasmodium falciparum (P. falciparum) erythrocyte membr ane protein 1 (PfEMP1) molecules. Results Our findings indicate that higher molecular mass (260 kDa-320 kDa) forms of Pf E MP1 were expressed on parasitized erythrocyte (PE) from human cerebral malaria p a tients. Compared with PfEMP1 expressed on PE from human cerebral malaria patien ts, the expression of PfEMP1 and Plasmodium vivax (P. vivax) erythrocyte me mbrane protein 1 (PvEMP1) on PE from falciparum malaria patients and vivax malar ia patients did not have multiple bands of PfEMP1 of ≥260 kDa, but had a PfEMP 1 with molecular mass of 240 kDa and a PvEMP1 with molecular mass of 180 kDa b and separately. Healthy controls expressed an EMP of molecular mass of 140 k Da.Conclusion Results confirm the antigenic variation of higher molecular mass of PfEMP1 whos e molecular mass is equal to or exceeds 260 kDa-320 kDa on PE of patients with cerebral malaria. Our results show that the binding of large antigenic variabi lity PfEMP1 molecular mass of 260 kDa-320 kDa on PE from human cerebral malari a patients with diverse receptor molecules on the endothelial cell (EC) of the c erebral microvessels may be involved in the molecular pathogenesis of cerebral m alaria.     

Immune responses to the expressed products of the CSP antige n gene of Plasmodium falciparum southern China isolate FCC1/HN in Hela cell

Objective To construct a eukaryotic expression system with pcDNA3-PfCSP/Hela for the Circ umsporozoite protein (CSP) gene of Plasmodium falciparum (P.falciparum), t o observe the immune responses in BALB/c mice induced by the expressed proteins .Methods The recombinant plasmid pcDNA3-PfCSP was transformed into the Hela cell line. The expressed protein was isolated and analyzed by using SDS-PAGE and used for immunization of BALB/c mice by subcutaneous, intravenous, and intraperitone al adminstration.Enzyme-linked immunosorbent assay(ELISA), Dot-ELISA, Wester n blot, T lymphocyte proliferation test, natural killer cell(NKC) activity assay , and CD4(+) and CD8(+) T cell detection were used for observation of humoral an d cellular immune responses.Results Immune sera strongly reacted with the expressed protein, antibody titer was up to 1∶6400 as detected by ELISA.Western blot analysis revealed a specific b and at 38.3 Kda.When the spleen cells of normal and immunized BALB/c mice we re specifically stimulated with expressed protein, the optical densities were 0 .12±0.03 and 0.34±0.04, respectively.The latter were significantly highe r than the former (P<0.01).We used the MTT colorimetric assay to measure NKC activity of mice spleen.The results showed that the NKC activity of immuni zed BALB/c mice was remarkably higher than that of the controls (P<0.05). CD4(+) and CD8(+) T cells were detected by using monoclonal antibody immunofluor escence methods.The results showed that the percentage of CD4(+) and CD8(+) T cells of immunized group were significantly higher than that of control group ( P<0.05).Conclusions The humoral and cell-mediated immune responses and elevated NKC activity to pr oducts made with a eukaryotic expression system could be specifically detected i n BALB/c mice.These findings indicate that the expressed protein could enhance the immune function in mice.     

Purification and Analysis of Soluble Antigens in Short Term Serum-Free in vitro Culture Medium of Plasmodium Falciparum

Supernatants collected from the short term in vitro serum-free cultures ofPlasmodium falciparum (Pf) schizont-infected erythrocytes were detected and analysed bycounterimmuno-electrophoresis (CIE) and enzyme-linked immunoabsorbent assay (ELISA).Soluble antigens were purified from the supernatants by immunoabsorbent chroma-tography. It was found that soluble antigens similar to the sonicated Pf antigens inimmunospecificity were present in the supernatants and no erythrocyte membrane antigenswere detectable in the affinity-purified supernatant antigens (Ps-Ag).