恶性疟原虫多表位重组疫苗在大肠杆菌中的表达及纯化

目的 在体外表达和纯化目的的蛋白,为下一步抗攻击试验提供安全有效的产品。方法 将化学合成的恶生疟原虫保护性抗原复合基因（ＨＧＦＳＰ）与表达载体ｐＲＳＥＴ重组,在大肠杆菌ＧＩ２１进行表达;工程菌经超声破菌、离心、离子交换层析、疏水层析、分子支析等步骤纯化。结果 ＳＤＳ－ＰＡＧＥ显示表达产物以非融合、可溶性的形式表达,相对分子质量为２３ｋＤａ,占总菌体蛋白的２３．６５％,纯度可达９５％以上。Ｗｅｓｔｅ     

Human phase I vaccine trials of 3 recombinant asexual stage malaria antigens with Montanide ISA720 adjuvant

Two phase I vaccine trials were conducted to test the immunogenicity and safety of a vaccine containing three recombinant malaria antigens from the asexual stage of Plasmodium falciparum. The three antigens are a fragment of MSP1 (190LCS.T3); MSP2 and a portion of RESA and were formulated in Montanide ISA720 adjuvant. These trials investigated the dose response of each antigen for eliciting both antibody and T-cell responses and the immunogenicity of a mixture of the antigens compared with the antigens injected separately. All three antigens elicited both antibody and T-cell responses. Strong T-cell responses were observed with 190LCS.T3 and RESA with stimulation indices exceeding 100 for peripheral blood leucocytes in some individuals. The antibody responses were generally weak. The human antibody responses observed with MSP2 in Montanide ISA720 were not significantly different from those obtained in an earlier trial which used MSP2 with alum as the adjuvant. No antigenic competition was observed: volunteers receiving a mixture of antigens had similar responses to those receiving the three antigens at separate sites. Tenderness and pain at the injection site were common over the first few days following immunization. In some volunteers, especially those receiving the highest doses tested, there was a delayed reaction at the injection site with pain and swelling occurring approximately 10 days after injection.     

Clinical efficacy and pharmacokinetics of artemisinin monotherapy and in combination with mefloquine in patients with falciparum malaria

1The aim of this study was to assess the pharmacokinetics, clinical efficacy and safety of artemisinin alone and in combination with mefloquine. 2Thirty-eight adults with symptomatic Plasmodium falciparum malaria were randomly assigned to receive either artemisinin (500 mg single dose followed by another 500 mg on day 1 and then 250 mg twice daily for 4 days) or artemisinin (500 mg single dose followed by 750 mg on day 1 and then 250 mg three times daily for one more day) in co-administration with mefloquine (250 mg three times daily for the first day). All drug administration was by the oral route. Patients were hospitalized at the Kibaha Designated District Hospital, Kibaha, Tanzania, for 6 days and a follow up for 3 weeks was performed. 3Treatment with the artemisinin/mefloquine combination resulted in a shorter parasite clearance time (PCT) of 24 (22, 27; 95% confidence interval) h vs 31 (27, 36) h and fever subsidence time (FST) of 14 (12, 16) h vs 20 (18, 23) h compared with artemisinin monotherapy. The 95% CI for the difference of the PCT and FST were 1.7, 12 and 3, 10, respectively. Parasites were detected in 7 out of 17 patients (41%) receiving artemisinin monotherapy at the 3rd and 4th week follow up visits. No parasites were detected after the combination therapy. 4The maximum plasma concentrations ( C_max) were similar after artemisinin monotherapy (615.4±387.0 ng ml⁻¹) and in combination with mefloquine (851.8±523.6 ng ml⁻¹). Elimination half-lives (t_1/2) were also identical at 2.2±0.6 h and 2.5±0.7 h, respectively. However, the AUC values were higher ( P<0.05) after combination therapy (3252±1873 ng  ml⁻¹ h) than after monotherapy (2234±1502 ng ml⁻¹ h). The oral clearance values were lower ( P<0.05) after combination therapy (195.4±86.9 l h⁻¹) than after monotherapy (314.3±189.4 l h⁻¹). PCT and FST normalized to initial parasitaemia correlated with AUC(0,  t) (r_s=0.56, P=0.02, r_s=0.58, P=0.01, respectively) and with C_max (r_s=0.62, P=0.01, r_s=0.68, P=0.005, respectively) in the artemisinin monotherapy only. 5One patient on the combination therapy developed a psychiatric condition and two patients on the monotherapy developed skin itch.     

Plasmodium vivax CS peptides display conformational preferences for folded forms in solution

Abstract: Spectroscopic techniques have been used to study the conformations of several synthetic peptides with sequences corresponding to the repeat regions of the circumsporozoite proteins of Plasmodium vivax, variants vk‐210 and vk‐247. As has previously been shown for P. falciparum, turn‐like folded conformations are observed, in rapid dynamic equilibrium with extended‐chain forms. These results are consistent with the known similarity of the structural, biosynthetic and immunological properties of the circumsporozoite proteins of different plasmodial species. Additionally, the observation of folded conformers provides a rationale for the effectiveness of these peptides as immunogens and potential vaccines.     

New Compounds Hybrids 1H‐1,2,3‐Triazole‐Quinoline Against Plasmodium falciparum

Malaria is one of the most prevalent parasitic diseases in the world. The global importance of this disease, current vector control limitations, and the absence of an effective vaccine make the use of therapeutic antimalarial drugs the main strategy to control malaria. Chloroquine is a cost‐effective antimalarial drug with a relatively robust safety profile, or therapeutic index. However, chloroquine is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of chloroquine‐resistant strains, which have also been reported for Plasmodium vivax. However, the activity of 1,2,3‐triazole derivatives against chloroquine‐sensitive and chloroquine‐resistant strains of P. falciparum has been reported in the literature. To enhance the anti‐P. falciparum activity of quinoline derivatives, we synthesized 11 new quinoline‐1H‐1,2,3‐triazole hybrids with different substituents in the 4‐positions of the 1H‐1,2,3‐triazole ring, which were assayed against the W2‐chloroquine‐resistant P. falciparum clone. Six compounds exhibited activity against the P. falciparum W2 clone, chloroquine‐resistant, with IC₅₀ values ranging from 1.4 to 46 μm . None of these compounds was toxic to a normal monkey kidney cell line, thus exhibiting good selectivity indexes, as high 351 for one compound ( 11 ).     

恶性疟原虫环子孢子蛋白在无细胞表达系统的表达及纯化研究

目的:应用麦胚无细胞表达系统(Wheat Germ Cell-Free System),表达及纯化恶性疟原虫环子孢子蛋白(Circumsporozo-ite protein,CSP),以便用于免疫学评价。方法:根据从PlasmoDB检索获得的恶性疟原虫CSP基因序列,设计一对特异引物,以从ATCC获得的恶性疟原虫3D7基因组质粒(P.f.3D7 genomic DNA)为模板,经PCR反应扩增目的片段。PCR产物经NcoI与Sma I双酶切后,与经过同样酶切的pIVEX 1.3WG载体连接,随后转化大肠杆菌DH5а,经筛选获得pIVEX WG1.3-CSP重组质粒。将该重组质粒加入到麦胚表达试剂盒并按照说明书进行表达,表达产物经亲和层析柱纯化,最终获得CSP蛋白。结果:PCR反应成功扩增CSP基因序列,并成功构建pIVEX WG1.3-CSP重组质粒;经WB试验证明,麦胚无细胞表达系统可成功表达并纯化CSP蛋白。结论:麦胚无细胞表达系统可高效表达恶性疟原虫CSP,为CSP用于后续的诊断试剂和疫苗评价方面的研究打下了坚实的基础。     

恶性疟原虫MESA基因的克隆和测序

目的克隆、测定恶性疟原虫海南株(FCC1／HN)成熟疟原虫感染红细胞表面抗原(MESA)基因序列，并进行序列分析。方法根据恶性疟原虫palo-alto株MESA基因已知序列，设计合成四对引物，用PCR技术从FCC1／HN株基因组DNA中扩增出4个部分序列重叠的MESA基因片段，分别克隆入pMD-18T测序载体。用双脱氧链末端终止法测定这4个基因片段的序列，拼接得到全长MESA基因序列。应用DNAstar、AnthProt软件辅助进行序列同源性比较和抗原表位区预测。结果PCR扩增得到特异的恶性疟原虫FCC1／HN株MESA基因片段，酶切及PCR鉴定获得了包含MESA基因片段的重组质粒。测序结果表明，FCC1／HN株MESA全基因编码区长4102bp，A T含量为72．11％，G C含量为27．89％，有1个内含子；编码1323个氨基酸残基，分子量为154470u。序列分析表明，FCC1／HN株与Palo-aho、D10株MESA蛋白在长度和序列组成上呈多态性，序列差异较大区域位于MESA蛋白的氨基酸重复区1、3、4、5和7。经多参数综合分析，有7个潜在的抗原表位区。结论测定、分析了恶性疟原虫FCC1／HN株MESA基因序列。FCC1／HN株MESA基因与其它分离株的MESA基因编码的氨基酸序列存在一定差异。     

一种简易疟原虫PCR模板制备方法

目的:寻找一种高效、简便、快速疟原虫PCR模板制备方法。方法:收集2010年-2011年自非洲、东南亚等疟疾流行区回国人员中疟疾现症患者(显微镜检查阳性),采滤纸血,用蒸馏水直接溶血法和Chel-ex-100法分别制备疟原虫PCR模板,采用巢式PCR技术检测疟原虫ss RNA基因,并对两法结果进行比较。结果:分别用蒸馏水直接溶血法和Chelex-100法制备疟原虫PCR模板32个,其中恶性疟原虫26个,间日疟原虫6个,直接溶血法巢式PCR检测全部出现特异性扩增带,而Chelex-100法均未出现扩增带。结论:蒸馏水直接溶血法是一种较为理想制备疟原虫PCR模板的方法。     

Molecular Detection of Plasmodium Infection among Anophelinae Mosquitoes and Differentiation of Biological Forms of Anopheles Stephensi Collected from Malarious Areas of Afghanistan and Iran

BackgroundUpdated information on the vectorial capacity of vectors is required in each malarious areas as well in Iran and its neighboring countries such as Afghanistan. The aims of this study were to investigate the potential infection of about 800 specimens collected from malarious areas of Afghanistan and Iran, and to differentiate biological forms of Anopheles stephensi.MethodTwo molecular markers, 18S RNA gene subunit and AsteObp1 intron I, were used respectively for investigation Plasmodium infection and identifying the biological forms of An. stephensi.ResultsPlasmodium infection was detected in 4 pools of Afghanistan specimens, including An. stephensi, collected from Nangarhar. Individually examination showed infection in 5 An. stephensi (infection rate: 1.25), to P. falciparum (2), P. vivax (2) and a mix infection. Out of five infected specimens, three were intermediate forms and two were mysorensis. No infection was found in specimens collected from Iran (Chabahar County), probably due to the active malaria control program in south-east of Iran.ConclusionThe key role of An. stephensi, as a known Asian malaria vector, was re-emphasized in Afghanistan by the results achieved here. The fauna of vectors and the pattern of biological forms of An. stephensi are similar in both countries that urge regional investigations to provide evidence-based and applied data for decision-maker in malaria control.     

疟疾双重PCR检测方法的建立和初步应用研究

目的 建立一种适合于我国恶性疟、间日疟和两混合感染地区,一次性检测人体内或蚊体内的恶性疟原虫(P.f.)和间日疟原虫(P.v,)的双重聚合酶链反应(PCR)法。方法 根据疟原虫红内期SSUrRNA基因特定片段,设计合成3条寡核苷酸特异引物,在一个反应体系中,同时检测P.f．与P．v.的双重PCR技术,操作方法按PCR常规。结果 从P.f.和P．v．感染血样中,分别扩增出274bp和412bp的特异片段,检出水平达1-5个虫／μl血,而人基因组DNA、约氏疟原虫、伯氏疟原虫均未见扩增条带;但食蟹猴疟原虫(P．c,)与P.v．相似,在412bp可见扩增条带。经对采自不同疟区发热待查病人的滤纸干血滴和确诊疟疾病人的静脉血检测,344例血样中,检出阴性112例、P.f．109例、P.v.l114例、P．f. P.v.9例,阳性率67．4％;比常规镜检的阳性率66．3％稍高,尤以检出P．f． P．v．感染病例高。分别检测人工感染P.f．与P.v.各10只大劣按蚊的阳性唾腺,均被检出同种的阳性结果。结论一次性检测P．f.DNA与P.v.DNA的双重PCR法,敏感性高,特异性强,稳定性好,具有简化操作、提高工效、降低耗费的优点。对发热待查病人诊断和蚊媒感染疟原虫调查,以及疟疾病原学的检测质控方面,均有实用价值。