环子孢子蛋白基因检测法对1例间日疟病例的溯源

对2014年1例初诊为河南省本地感染的间日疟病例进行实验室检测与流行病学溯源调查,以明确其感染来源。收集该病例的流行病学资料,分别采用厚薄血膜吉氏染色法、疟疾快速诊断试纸(RDT)法和巢式PCR法检查患者外周血液,并对其环子孢子蛋白(CSP)基因序列进行分析。流行病学调查显示,该患者曾于2013年5月至缅甸停留约1周,同年6月发病,确诊为间日疟,经青蒿琥酯治疗后,疟原虫转阴,症状消失。CSP基因序列分析显示,该患者前后两次发病时的血样扩增出的CSP序列的中央重复区完全一致,与缅甸分离株(Gen Bank登录号为ABS95455和ABS95456)的序列一致性分别为95.1%和100%,与2个河南分离株HN3和HN7(登录号为KP888996和KP889000)的序列一致性分别为88.8%和67.1%。推测该病例为输入性间日疟复发病例。     

间日疟原虫深圳株红内期SSUrDNA基因片段的克隆与序列分析

目的 体外扩增间日疟原虫深圳株红内期小亚单位核糖体核糖核酸编码基因(SSUrDNA)片段，研究其结构与功能。方法 设计一对特异性引物，采用聚合酶链反应(PCR)从间日疟原虫患者血样中扩增出间日疟原虫SSUrDNA片段，以PUC19质粒T载体构建重组子导入大肠杆菌JM109；阳性克隆双酶切鉴定后，双脱氧末端终止法测定序列。结果 间日疟原虫SSUrDNA扩增片段大小为341bp；阳性克隆双酶切及PCR扩增均得到预期大小的片段；序列测定插入片段为341bp，与Sal I株顺序相比，仅在第151位处缺失一个碱基C。结论 成功克隆了间日疟原虫SSUrDNA片段．该序列在间日疟原虫虫株间高度保守。     

长沙、上海、郑州、武汉中华按蚊对慈利、沔阳间日疟原虫易感性的交叉对比

来自不同疟疾疫区——长沙、上海、郑州、武汉四地区的中华按蚊,分别以羊膜饲血法感染湖南慈利或湖北沔阳两地区的间日疟原虫,交叉对比其易感性。七次实验共解剖饲血蚊541只。实验结果显示:以腺感染率和阳性腺指数为主要指标,四地区中华按蚊对间日疟原虫的易感性基本相似:两地区间日疟原虫对中华按蚊的感染力也大致相同。并讨论了可能影响人工感染率的几个因素。     

The Receptor Role of Glycophorin A during the Invasion of Erythrocytes by Plasmodium Falciparum

Under the condition of in vitro culture,the effects of glycophorin A(GPA)of human erythrocyte membrane,anti-GPA IgG,α_1-acid glycoprotein,ovomucoid,wheat germ agglutinin,etc.on the invasion of erythrocytes by P.falciparum were observed in this study.The results show that the combination ofGPA with P.falciparum merozoites has high specificity,high affinity and atendency to saturation,and produces specific biological effects.These confirm forthe first time from the characteristics of the ligand-receptor interaction that GPAis a receptor recognized and bound by P.falciparum merozoites.     

对间日疟原虫配子体活力消长的进一步观察

以常用的体外吸血法,对2例人工接种大量子孢子、发病后未用抗疟药治疗的间日疟现症病例,每天在发作间歇期,取静脉血感染驯化的中华按蚊,直至原虫密度下降,病程自然结束为止。结果表明,间日疟病例在临床发作的第1天对按蚊即有感染性,感染高峰见于第8病日之前,自第9病日显著下降。高感染度的持续时间极短,1例为3天,另1例为4天,分别见于发病的第4~6天和第3~6天,相当于1.5~3个无性体裂体增殖周期,以后一直维持在很低水平,且无上升趋势。由此可见,早期发现和及时治疗,在疟疾防治工作中具有重要意义;在科研工作中,凡需大量子孢子进行实验研究时,选择发作不超过3次、原虫血症较高的早期现症病例作为传染源最为适宜。     

PCR检测食蟹猴疟原虫的实验研究

目的 建立一种特异、敏感、简捷的PCR检测食蟹猴疟原虫（P.C）的方法。方法 检测样品来自人工接种感染P.C的阳性猴血膜及滤纸血斑。根据P.C　SSUrRNA基因序列,设计P.C特异引物Pc1和Pc3,用PCR常规操作对P.C DNA模板扩增。同时用多对间日疟原虫（P.V）特异引物对照比较。结果 从 P.C血样中扩增出 425bp特定扩增带,而P.V红内期和子孢子、恶性疟原虫、伯氏疟原虫、人基因组DNA和健康猴血均未见扩增带;敏感度可检测 1.68 ×10-6的原虫感染水平。同时发现P.C对多对 P.V特异引物均出现阳性扩增带,以往一直未被注意。结论 该方法检测 P.C特异、敏感和简捷,提供了鉴别 P.V与 P.C的准确诊断。建议在 PCR的P.V引物设计和特异性的检测过程,把应用P.C　DNA检测列为常规对照,以提高检测质量。     

复方萘酚喹治疗间日疟的疗效观察

目的 :观察复方萘酚喹与氯喹治疗间日疟的临床疗效。方法 :以显微镜血检单纯间日疟原虫阳性患者为观察对象 ,药物为复方萘酚喹 (萘酚喹 40 0mg 青蒿素 1 0 0 0mg) ,1次顿服 ,服药后按时测量体温和血检原虫 ,随访 42天 ,观察治疗效果。结果 :复方萘酚喹治疗 1 0 9例 ,平均退热时间为 ( 1 3 0± 5 5 )h ,原虫转阴时间为 ( 1 8 1± 5 7)h,治愈率为 1 0 0 %。药后无明显不良反应。结论 :复方萘酚喹治疗间日疟具有良好的效果。     

一氧化氮对红内期约氏疟原虫感染的影响

目的：探讨的氏疟原虫感染早期一氧化氮（nitric oxide,NO)对红内期疟原虫的影响。方法：通过ELISA和Griess反应,观察疟原虫感染早期宿主体内细胞因子和NO对红内期虫体血症的影响及其效应。结果：疟原虫感染早期随着以IFN-γ分泌增加为主的Th1细胞免疫应答产生的过程,NO合成逐渐增加,红内期原虫血症受到抑制,一旦解除高水平NO的作用,虫体的增殖能力即可迅速得以恢复。结论：NO通过抑制裂殖子的侵袭力发挥抗红内期原虫感染的保护性免疫效应。     

Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax.

Molecular cloning and structure analysis of the gene encoding the Pv200 protein of the Sal-1 strain of Plasmodium vivax revealed an overall identity of 34–37% when the deduced amino acid sequence was compared with the sequences of various major merozoite surface antigens of Plasmodium falciparum, Plasmodium yoelii and Plasmodium chabaudi. When the Sal-1 Pv200 sequence was compared with the corresponding sequence from the Belèm strain of P. vivax, it was found that the two merozoite surface antigens were relatively well conserved with an overall amino acid sequence identity of 81%. A region of 23 repeated glutamine residues, found in the sequence of the Belèm isolate was not found, however, in the Sal-1 sequence. Amino-and carboxy-terminal domains of the Pv200 protein were expressed in the yeast Saccharomyces cerevisiae. Each recombinant protein was shown to react with antibodies in sera from splenectomized Bolivian Saimiri monkeys that had been infected previously with P. vivax, and in human sera from individuals with a history of exposure to vivax malaria. The availability of recombinant DNA-derived Pv200 proteins will now allow a full assessment of their utility in the diagnosis and immunoprophylaxis of the benign tertian malaria associated with P. vivax infection.     

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.