间日疟原虫和恶性疟原虫可溶性抗原免疫反应性比较

目的分析和比较间日疟原虫(Plasmodiumvivax,P.v)和恶性疟原虫(P.falciparum,P.f)可溶性抗原与间日疟感染者混合血清和单克隆抗体(McAb)M26-32免疫反应性的差别,以期寻找潜在的疟疾诊断抗原。方法取P.v感染者血样,用Plasmodipur滤器分离去除白细胞,经60%Percoll浓集其中的感染红细胞(i RBC)。分别制备P.v、P.f和正常红细胞(nRBC)可溶性抗原,应用P.v感染者、正常对照混合血清和M26-32单抗与相应的抗原进行免疫印迹分析。结果免疫印迹分析表明,P.v感染者能特异性识别26k、33、49、115kDaP.v抗原;100、102、110、150、175kDaP.f抗原;能够交叉识别的条带有:31、59、63、70、120kDa。M26-32单抗能识别P.f、P.v抗原中31kDa等抗原条带。结论P.v感染者能特异识别间日疟抗原组份,并能交叉识别P.f抗原,其中31kDa抗原组分具有较强的免疫原性,同时能被M26-32单抗识别,其作为P.v特异性诊断抗原的价值有待于进一步研究。     

HIV p24抗原的检测及意义

艾滋病 (ＡｃｑｕｉｒｅｄＩｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙＳｙｎｄｒｏｍｅ ,ＡＩＤＳ)在全球广泛流行 ,亚洲是继北美、非洲之后成为全球ＨＩＶ (ＨｕｍａｎＩｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙＶｉｒｕｓ)感染上升最迅速、流行最严重的地区之一。我国ＡＩＤＳ也呈加速流行的趋势 ,截止到 2 0 0 0年 9月 ,共报告ＨＩＶ感染者 2 0 711例。据专家估算 ,我国实际感染人数超过 60万人。ＨＩＶ主要通过性、母婴和血液三种途径传播。为监控流行情况 ,评价新的疫苗和药物 ,建立简便、灵敏、特异的检测方法特别关键。理想的检测试剂应该是拥有一套…     

类鼻疽菌特异性抗原的制备与应用—报道Ⅱ

本实验采用菌体吸收和亲和层折技术,对类鼻疽一次亲和抗原进一步纯化,获得了类鼻疽两次亲和抗原。经SDS-PAGE和等电聚焦电泳测定,该抗原分子量为15,100,至少由两个蛋白值亚基组成,其等电点分别为5.32和5.54;该抗原圆盘电泳薄层扫描纯度为91.9％;与一次亲和抗原比较,该抗原与马鼻疽的交叉至少减少4倍;浓度为100ng/ml的该抗原在放射免疫测定中活性高,并且不与马鼻疽出现交叉,在临床血清标本的试测中收到了较好的效果。用该抗原制备的免疫兔血清在琼脂双向扩散中与马鼻疽抗原不出现沉淀线,与类鼻疽粗提抗原仅有一条沉淀线,显示了较高的特异性。     

含有弓形虫表面抗原P22、P30复合基因的载体构建

目的　选择弓形虫表面抗原P2 2、P30的有效基因片段 ,共同构建在同一克隆载体pUC19和表达载体 pGE MEXTM- 1上 ,并保证其连接方向及开放读码框的正确。方法　根据已发表P30基因序列 ,用PCR技术调取所需基因片段 ,P2 2基因片段来自重组质粒pGEX - 2T -v2 2 ,将两片段定向克隆于克隆载体 pUC19及表达质粒pGEMEXTM- 1上 ,用酶切及测序的方法对重组子进行鉴定。结果　酶切产物经电泳显示条带清晰 ,P2 2、P30基因片段的泳动位置分别在 4 4 6bp和 86 0bp的位置 ,与预计结果一致 ;测序结果表明插入的复合基因片段方向及序列均正确。结论　成功构建的含有P2 2、P30基因片段的质粒重组体 ,保证了两基因连接方向、序列及开放读码框的正确 ,为今后两基因的进一步复合表达研究奠定了基础。     

P53 accumulation and proliferating-cell nuclear antigen expression in human lung cancer

Mutations in thep53 gene are currently the commonest genetic alterations in human malignant tumors, including non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Alterations of the protein induced by gene mutations enables the mutant protein to become more stable, resulting in the accumulation of P53 in quantities detectable by immunohistochemistry. Although previous studies document the accumulation of P53 in lung cancer, there is little information regarding the usual frequency of accumulation based on a comprehensive number of lung tumors. A total of 328 paraffin-embedded lung carcinoma specimens were analyzed for P53 accumulation and for the expression of the proliferating-cell nuclear antigen (PCNA) by standard immunohistochemistry. Among 49 SCLC, 35% were positive for p53 and 51% were positive for PCNA. Out of 279 NSCLC, 43% showed a positive P53 immunoreaction and 72% displayed detectable amounts of PCNA. In squamous-cell carcinomas a statistically significant increased accumulation of P53 was found compared to adenocarcinomas (P=0.001). Among the 233 PCNA-positive tumors the relative number of P53-positive specimens was higher compared to the total number of tumors. Since immunohistochemical investigations should contribute to the improvement of the clinical diagnosis and treatment or give information on the prognosis, we conclude from our results that it seems to be legitimate to assess the P53 status exclusively in the specimens positive for PCNA. Immunohistochemical investigations under consideration of the PCNA status yielded good and fast recognition ofp53 mutations leading to intracellular P53 protein accumulation.Abbreviations SCLC small-cell carcinoma - NSCLC non-small-cell carcinoma - PCNA proliferating-cell nuclear antigen     

弓形虫RH株膜蛋白P30的原核表达与鉴定

目的在E.coli中表达弓形虫RH株膜蛋白P30。方法将P30基因定向克隆到pET28b,构建含目的基因的pET28b-P30重组质粒,转化E.coliBL21(DE3),接种含pET28b-P30的BL21(DE3)单菌落于LB培养基,1∶100稀释后用0.2mmol/L的IPTG诱导表达,SDS-PAGE和Westernblot鉴定诱导表达产物。结果1构建了重组质粒pET28b-P30,2在E.coli中表达了一分子量约为30kDa的融合蛋白,经Westernblot鉴定正确。结论在E.coli中高效表达了弓形虫RH株膜蛋白P30,并以包涵体形式存在。     

Proliferating cell nuclear antigen and P53 protein expression and prognosis in primary liver cancer

AIM: To study the individual difference and prognosis of primary liver cancer (PLC) after hepatectomy. METHODS: Two hundred specimens obtained from PLC patients were examined immunohistochemically using anti-P53 and anti-PCNA monoclonal antibodies. The mutations of p53 in exons 5-8 of the gene were examined using the polymerase chain reaction/single-stranded conformational polymorphism (PCR/SSCP) method in 60 of the 200 specimens. RESULTS: The results showed that the labeling index of proliferating cell nuclear antigen (PCNA) and expression of p53 had a parallel correlation (P < 0.001). The high labeling index of PCNA or overexpression of p53 was associated with a high risk of tumor recurrence, more aggressive growth and poor survival. CONCLUSION: Immunohistochemical assessment of PCNA and P53 can provide very useful information for the prognosis of PLC.     

弓形虫昆山分离株P30蛋白的纯化、复性和鉴定

目的表达并纯化弓形虫昆山分离株膜蛋白P30。方法将构建的pET28b-P30重组质粒转入大肠埃希菌BL21,挑重组菌于LB培养基中增菌,IPTG诱导其表达,然后纯化包涵体,用尿素溶解包涵体,Ni2 螯合柱纯化产物,以透析法复性蛋白,并用SDS-PAGE和Western blot进行鉴定。结果大肠埃希菌表达的P30蛋白以包涵体的形式存在,P30蛋白经Ni2 螯合柱纯化后纯度大于95%。经Western blot鉴定,复性蛋白能被弓形虫感染的鼠血清识别。结论得到了纯度高的膜蛋白P30。     

人工合成三糖抗原在结核病血清诊断中的初步评价

目的评价人工合成三糖抗原在结核病血清诊断的应用前景?方法用人工合成的结核杆菌酚糖酯三糖抗原Tb NT P BSA(naturaltrisaccharide phenylpropionate bovineserumalbumin)作抗原,用ELISA法检测了40例结核患者,52个健康对照的血清标本?结果本方法的特异性?敏感性分别为:检测抗Tb NT P BSAIgG抗体时为94.2%和20.0%;检测抗Tb NT P BSAIgM抗体时为79.6%和45.0%?结论该抗原的特异性?敏感性似不理想,有待进一步改进。     

Cross-reacting antigens to Pc96, a protective antigen of Plasmodium chabaudi, in P. falciparum, P. vivax, and P. cynomolgi

Mice can be partially protected against Plasmodium chabaudi by immunization with the antigen Pc96, isolated from the erythrocyte membranes of infected mice. We used a Pc96 specific monoclonal antibody to identify antigens which cross-react with Pc96 in P. falciparum, P. vivax, and P. cynomologi. The cross-reactive molecules are antigens of Mr 155,000 in P. falciparum, Mr 220,000 in P. cynomologi. They are located in the surface membranes of infected erythrocytes. Pc96 is characterized by immunoelectron microscopy and epitope mapping.     

结直肠癌手术前后血清P53抗体,CEA及CA19-9的检测意义

目的：通过检测结直肠癌患者手术前后血清P53抗体,CEA,CA19-9的变化,评价其临床意义．方法：选择2002-04/2003-01手术治疗的结直肠癌患者132例和36例非肿瘤患者为研究对象．用ELISA法定性检测血清P53抗体,放射免疫分析法(RIA)定量检测CEA,CA19-9．结果：结直肠癌组中53例血清P53抗体阳性,而对照组仅1例阳性(X~2=18．11,P＜0．0001)．CEA和CA19-9在结直肠癌患者中的阳性率分别为18．2％和12．9％．血清P53抗体阳性患者术后1mo有39例(74％)抗体转阴,其中姑息性手术组3例(33％),而根治性切除组36例(82％)(X~2=9．04,P=0．0026)．CEA阳性的24例患者中,术后1 mo有13例(54％)转为阴性,其中姑息性手术组2例(18％),而根治性手术组11例(85％)(X~2=10．60,P=0．0011)．术前CA19-9阳性的17例患者中7例(41％)在术后1mo转为阴性,在姑息性手术组和根治性切除组的转阴率分别为20％(2例)和71％(5例)(X~2=4．50,P= 0．034)．结论：血清P53抗体,CEA,CA19-9的手术前后动态检测有助于临床判定病情,监测疗效．     

弓形虫表面抗原P22蛋白的研究进展

*　弓形虫(Toxoplasma gondii)是专性寄生于人和多种动物组织有核细胞内的原虫,呈世界性分布。近年来关于弓形虫细胞表面抗原作为诊断试剂及免疫疫苗的双重潜在价值得到广泛研究,主要发现有5种表面抗原:P22、P23、P30、P35、P43。其中P22基因依据克隆的顺序也被称为SAG2。本文就弓形虫重要抗原P22的基因和蛋白结构,及该抗原在弓形虫虫株的分型、血清诊断、疫苗方面的研究作简要介绍。1　关于弓形虫P22基因弓形虫的生活史中除了合子期外,其他期的核均为单倍体。已经确定的染色体有8条, 各编码染色体分子质量单位分别为: 2.0、2.4、2.6、…     

Sensitivity of P. vivax rapid antigen detection tests and possible implications for self-diagnostic use

In a prospective study amongst febrile travellers returning from malaria-endemic areas to Berlin, Germany, two rapid malarial antigen detection tests were compared for the diagnosis of vivax malaria with routine microscopy. With ICT Malaria P.f./P.v.((R)), 664 samples of 492 patients were examined. 17 patients had vivax malaria, out of which 11 infections were missed (35.3% sensitivity). With OptiMal((R)), 659 samples of 539 patients were examined. 22 patients had vivax malaria, and all infections were identified correctly (100% sensitivity). Specificity was 100% with both tests. The ICT Malaria P.f./P.v.((R)) is advertised for layman use during travel, and the literature was reviewed with respect to the question of suitability of these devices for self-testing. It is concluded that with the ICT Malaria P.f./P.v.((R)), the detection of non-falciparum (i.e. predominantly vivax) malaria is unreliable, and test interpretation for medically untrained individuals particularly in distress might be too complicated even after proper instruction.     

旋毛虫ES抗原P49基因的体外表达分析

目的克隆和表达旋毛虫ES抗原P49基因,并分析表达产物的免疫原性。方法以重组质粒pCDNA3.1 P49为模板,PCR扩增旋毛虫ES抗原P49基因,将扩增片段双酶切产物连接到质粒pEGFP N1中,构建重组真核表达载体pEGFP N1 P49,利用脂质体法将其转染哺乳动物细胞COS 7。于转染24h后在荧光显微镜下观察发绿色荧光的转基因细胞;通过Western blot分析证实P49基因表达产物的免疫原性。结果成功构建旋毛虫ES抗原P49基因的绿色荧光蛋白表达载体pEGFP N1 P49,并在COS 7细胞中表达了目的蛋白,并且表达的蛋白可被感染旋毛虫小鼠阳性血清特异地识别。结论成功获得了重组蛋白,并证实该融合蛋白具有免疫原性,其结果为研究其生物学功能奠定了基础。     

Antibodies to Plasmodium falciparum ring-infected erythrocyte surface antigen and P. falciparum and P. malariae circumsporozoite proteins: seasonal prevalence in Kenyan villages

Two cross-sectional surveys of 954 persons in Asembo Bay and Got Nyabondo, western Kenya, were performed in August-September 1986, after long rains, and in February-March 1987, after a comparatively dry season. Serologic testing was performed using an ELISA with synthetic peptides representing repeat amino acid sequences of the Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA), (EENV)5, (EENVEHDA)4, and (DDEHVEEPTVA)2 and repeat sequences (PNAN)5 and (NAAG)5 of the P. falciparum and P. malariae circumsporozoite proteins. In 1986, 45%, 73%, 72%, 85%, and 59% of the persons in Asembo Bay had antibodies to the respective peptides. In Got Nyabondo, the rates were 44%, 67%, 56%, 36%, and 41%, respectively. All positivity rates increased with age. When next determined in 1987, the positivity rates and levels of reactivity were generally unchanged in Asembo Bay, but were decreased in Got Nyabondo.     

T cell-mediated immunity in murine malaria. II. Induction of protective immunity to P. chabaudi by antigen fed macrophages and antigen educated lymphocytes

We previously described assay systems for generating antigen specific proliferating T cells to P. chabaudi antigens. In the present study we examine whether the various sensitization approaches confer immunity against a cloned virulent strain IP-PCI of P. chabaudi. We present data indicating that effective specific protective immunity can be induced through P. chabaudi antigen fed macrophages and antigen educated spleen cells (initiator lymphocytes). The expression of this protective immunity is proposed to depend on (a) antigen presentation and/or accessory function of macrophages and (b) the subsequent activation of T cell functions related to protection. Indeed analysis of different macrophage populations revealed a correlation between the expression of Ia molecules and IL-1 secretion with their capacity to induce antigen specific T cells in vivo and subsequent protective immune mechanisms. Thus these results emphasize the critical functions of accessory cells in determining the outcome of malaria infections.     

Ribosomal P protein P0 as a candidate for the target antigen of anti-endothelial cell antibodies in mixed connective tissue disease

OBJECTIVE: To evaluate the presence of anti-endothelial cell antibodies (AECA) in patients with mixed connective tissue disease (MCTD) compared to those with systemic sclerosis (SSc) and to determine the candidates for the endothelial auto-antigen that reacts with AECA in patients with MCTD using a molecular cloning strategy. METHODS: AECA were measured by a cellular enzyme-linked immunosorbent assay (ELISA) using fixed human umbilical vein endothelial cells (HUVEC) in 47 MCTD patients, 68 SSc patients, and 52 normal controls. A HUVEC cDNA expression library was immunoscreened with pooled sera from 6 patients with high AECA levels determined by cellular ELISA to explore the endothelial autoantigens in MCTD. An ELISA assay for anti-ribosomal protein P0 antibodies was used to assess the correlation with AECA levels. RESULTS: The candidate target proteins recognized by AECA in MCTD included: (i) ribosomal protein P0; (ii) a putative oncogene derived from dek mRNA; (iii) SS-B/La protein; (iv) U1 RNA-associated 70K protein; and (v) DNA-binding protein B. A significant correlation between the levels of AECA and anti-ribosomal protein P0 antibodies was demon-strated in MCTD, but not in systemic sclerosis. The sera containing high levels of AECA from patients with MCTD frequently cross-reacted with ribosomal protein P0. On the other hand, sera without AECA activity from patients with MCTD never reacted with ribosomal protein P0. CONCLUSION: AECA were more frequently seen in patients with MCTD than in patients with SSc. Ribosomal protein P0 may be one of the major target antigens of AECA in patients with MCTD.     

噬菌体展示技术筛选人隐孢子虫P23抗原表位

目的本研究利用噬菌体展示技术筛选人隐孢子虫P23抗原表位,为研制其抗原表位疫苗奠定基础。方法首先用人隐孢子虫重组P23抗原免疫动物以制备抗体,将该抗体与噬菌体展示随机肽库结合,通过ELISA和Western-blot方法鉴定阳性克隆,并对阳性克隆进行增殖,然后对其序列测定和抗原表位预测。结果噬菌体肽库结合抗体后,经ELISA方法可鉴定10株为阳性克隆,Western-blot检测可发现有67kD目的条带出现;序列分析结果可发现它们中具有3个不同结构表位序列,命名为ep1,ep2,ep3,且预测出具有良好的亲水性。结论所得序列是具有B细胞抗原表位。