弓形虫GRA6可溶性蛋白在大肠埃希菌中的表达及纯化

目的在大肠埃希菌中高效表达及纯化弓形虫GRA6抗原,为制作弓形虫感染基因工程诊断试剂盒奠定基础.方法将重组pGEX-GRA6表达载体转化大肠埃希菌BL21-Codon Plus（DE3）-RP菌株,在异丙基硫代-β-D半乳糖苷（IPTG）诱导下表达.超声破壁后,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表达产物的表达形式,并对表达产物以硫酸铵沉淀、Sephedax G50脱盐和GST亲和层析柱进行目的蛋白的纯化.通过Western blotting检测其纯化的重组抗原的免疫反应性.结果GRA6以融合蛋白（GST-GRA6）的形式在大肠埃希菌中得到了高效表达.对表达产物可溶性分析表明,表达蛋白在上清液中和包涵体中均有表达.在上清液中表达的可溶性蛋白经纯化后蛋白纯度可达90%以上.Western blotting分析表明纯化蛋白能被弓形虫感染的人血清所识别.结论GRA6在大肠埃希菌中得到了高效表达,重组抗原经纯化后能特异性地被弓形虫感染者血清所识别.     

外源性一氧化氮对弓形虫速殖子凋亡的诱导作用

目的　探讨一氧化氮 (NO)是否能诱导弓形虫速殖子凋亡。方法　采用末端脱氧核苷酸转移酶(TdT)介导的原位末端标记法 (TUNEL)、透射电镜和琼脂糖凝胶电泳检测弓形虫速殖子凋亡的特征。结果　TUNEL标记法检测表明 ,NO供体亚硝基铁氰化钠 (SNP)可诱导弓形虫速殖子凋亡 ,并呈剂量和时间依赖性 ;NO清除剂 ,N 乙酰半胱氨酸能明显抑制SNP诱导的弓形虫速殖子凋亡 ;不含NO的SNP类似物 ,铁氰化钾不能诱导弓形虫速殖子凋亡。透射电镜下见SNP处理 15～ 2 0h的速殖子具有凋亡的典型形态学特征 :核染色质凝集、核固缩、核碎裂和凋亡小体形成。琼脂糖凝胶电泳显示SNP处理的弓形虫速殖子DNA片段呈现凋亡特征性的梯形条带。结论　从形态学和生化特征证明由SNP释出的NO可诱导弓形虫速殖子凋亡     

Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA–RON2 pair

Plasmodium falciparum and Toxoplasma gondii are widely studied parasites in phylum Apicomplexa and the etiological agents of severe human malaria and toxoplasmosis, respectively. These intracellular pathogens have evolved a sophisticated invasion strategy that relies on delivery of proteins into the host cell, where parasite-derived rhoptry neck protein 2 (RON2) family members localize to the host outer membrane and serve as ligands for apical membrane antigen (AMA) family surface proteins displayed on the parasite. Recently, we showed that T. gondii harbors a novel AMA designated as TgAMA4 that shows extreme sequence divergence from all characterized AMA family members. Here we show that sporozoite-expressed TgAMA4 clusters in a distinct phylogenetic clade with Plasmodium merozoite apical erythrocyte-binding ligand (MAEBL) proteins and forms a high-affinity, functional complex with its coevolved partner, TgRON2_L1. High-resolution crystal structures of TgAMA4 in the apo and TgRON2_L1-bound forms complemented with alanine scanning mutagenesis data reveal an unexpected architecture and assembly mechanism relative to previously characterized AMA–RON2 complexes. Principally, TgAMA4 lacks both a deep surface groove and a key surface loop that have been established to govern RON2 ligand binding selectivity in other AMAs. Our study reveals a previously underappreciated level of molecular diversity at the parasite–host-cell interface and offers intriguing insight into the adaptation strategies underlying sporozoite invasion. Moreover, our data offer the potential for improved design of neutralizing therapeutics targeting a broad range of AMA–RON2 pairs and apicomplexan invasive stages.Phylum Apicomplexa comprises >5,000 parasitic protozoan species, many of which cause devastating diseases on a global scale. Two of the most prevalent species are Toxoplasma gondii and Plasmodium falciparum, the causative agents of toxoplasmosis and severe human malaria, respectively (1, 2). The obligate intracellular apicomplexan parasites lead complex and diverse lifestyles that require invasion of many different cell types. Despite this diversity of target host cells, most apicomplexans maintain a generally conserved mechanism for active invasion (3). The parasite initially glides over the surface of a host cell and then reorients to place its apical end in close contact to the host-cell membrane. After this initial attachment, a circumferential ring of adhesion (termed the moving or tight junction) is formed, through which the parasite actively propels itself while concurrently depressing the host-cell membrane to create a nascent protective vacuole (4).Formation of the moving junction relies on a pair of highly conserved parasite proteins: rhoptry neck protein 2 (RON2) and apical membrane antigen 1 (AMA1). Initially, parasites discharge RON2 into the host cell membrane where an extracellular portion (domain 3; D3) serves as a ligand for AMA1 displayed on the parasite surface (5–8). Intriguingly, recent studies have shown that the AMA1–RON2 complex is an attractive target for therapeutic intervention (9–12). The importance of the AMA1–RON2 pairing is also reflected in the observation that many apicomplexan parasites encode functional paralogs that are generally expressed in a stage-specific manner (13–15). We recently showed that, in addition to AMA1 and RON2, T. gondii harbors three additional AMA paralogs and two additional RON2 paralogs (14, 15): TgAMA2 forms a functional invasion complex with TgRON2 (15), TgAMA3 (also annotated as SporoAMA1) selectively coordinates TgRON2_L2 (14), and TgAMA4 binds TgRON2_L1 (15). Despite substantial sequence divergence, structural characterization of all AMA–RON2D3 complexes solved to date [TgAMA1–TgRON2D3 (16), PfAMA1–PfRON2D3 (17), and TgAMA3–TgRON2_L2D3 (14)] reveal a largely conserved architecture and binding paradigm. Intriguingly, however, sequence analysis indicates that TgAMA4 and TgRON2_L1 are likely to adopt substantially divergent structures with an atypical assembly mechanism.To investigate the functional implications of the AMA4–RON2_L1 complex in T. gondii, we first established that TgAMA4 is part of a highly divergent AMA clade that includes the functionally important malaria vaccine candidate Plasmodium merozoite apical erythrocyte-binding ligand (MAEBL) (18–20) and that TgRON2_L1 displays a similar divergence consistent with coevolution of receptor and ligand. We then show that TgAMA4 and TgRON2_L1 form a high-affinity binary complex and probe its overall architecture and underlying mechanism of assembly using crystal structures of TgAMA4 in the apo and TgRON2_L1D3 bound forms. Finally, we show proof of principle that TgAMA4 and TgRON2_L1 form a functional pairing capable of supporting host-cell invasion. Collectively, our study reveals exceptional molecular diversity at the parasite–host-cell interface that we discuss in the context of the unique invasion barriers encountered by the sporozoite.     

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。     

刚地弓形虫P30(SAG1)基因的克隆与表达

目的构建弓形虫P30基因表达载体并获得重组表达蛋白。方法将弓形虫P30基因的开读框用PCR扩增,NcoⅠ和HindⅢ酶切后,与同样酶切的表达质粒pET30a(+)经T4连接酶连接,然后转化到DH5α中。菌液经PCR扩增和质粒酶切及基因测序鉴定后,将阳性重组质粒转化到大肠埃希菌BL21(DE3)中,经IPTG诱导,表达产物用SDS PAGE和Westernblot进行鉴定。结果扩增的P30基因片段为750bp,重组质粒诱导表达产物分子质量单位为30ku,与理论值相符。Westernblot确认重组质粒表达蛋白与小鼠抗弓形虫单克隆抗体(P30McAb)发生特异性反应。结论成功构建重组体并获得弓形虫主要表面抗原P30的高效表达产物,为弓形虫病的诊断和疫苗研究奠定了基础。     

弓形虫感染对雄性小鼠睾丸细胞周期影响的实验研究

目的　研究弓形虫感染对雄性小鼠睾丸组织细胞周期的影响。方法　应用流式细胞术检测弓形虫感染10 3/ ml、10 4 / ml、10 6 / ml3个剂量组小鼠睾丸组织细胞周期,并设立生理盐水和环磷酰胺对照组。结果　不同剂量的弓形虫感染均可使小鼠睾丸组织细胞各时相的细胞百分数发生变化,并随着剂量的增加,G0 / G1 、S时相的细胞百分数明显减少,与阴性对照组比较差异有显著性(P<0 .0 5 )。G2 / M时相的细胞百分数逐渐增多,各剂量组与阴性对照组比较差异有显著性(P<0 .0 5 )。结论　弓形虫感染可能抑制小鼠睾丸细胞的DNA合成,可引起G2 期细胞阻滞,从而使细胞的有丝分裂延伸。     

弓形虫表面抗原研究进展

刚地弓形虫是一种广泛分布于人和哺乳动物组织细胞内的机会性致病原虫,可引起严重的人兽共患病.弓形虫抗原的研究对开发诊断制剂、疫苗以及从分子水平探讨弓形虫与宿主之间的相互关系具有重要意义,该文对近年来有关弓形虫抗原的研究进展作一综述.     

应用DIFA等三项试验联合筛选孕妇弓形虫感染的研究

用DIFA初筛检测197例武汉地区非选择性孕妇，其弓形虫抗体阳性率为8．12％（16／197）；阳性者用IFT—IgG复查，阳性符合率为93．75％，即阳性率为7.61％（15／197），15例阳性者的滴度分别为1：164例，1:649例，≥1：10242例；DIFA阳性者还用Toxo－IgG／IgA／IgM－ISAGA检测，有2例IgA和IgM均为阳性。其中1例IgA1：64（＋）、IgM1：16384（＋）为急性弓形虫感染件发葡萄胎。DIFA是一种敏感性高、特异性强、操作简便适用于孕妇弓形虫感染初筛的好方法，为达到优生优育监测弓形虫感染的目的，而采用DIFA、IFT－IgG、Toxo－IgG／IgA／IgM联合测试是较好的方案。     

刚地弓形虫ROP2-P30复合重组蛋白疫苗对BALB／c小鼠免疫保护性的研究

目的用刚地弓形虫ROP2-P30复合重组蛋白疫苗免疫BALB/c小鼠观察对机体的影响,为研制安全有效的弓形虫疫苗奠定基础。方法PCR方法从刚地弓形虫基因组中扩增出ROP2和P30片段,将这两个片段分别亚克隆至pET-30a原核表达载体中,表达出ROP2-P30复合重组蛋白。用蛋白疫苗免疫BALB/c小鼠,ELISA检测免疫小鼠后体液中抗体和细胞因子的变化,观察攻击试验小鼠的的死亡率。结果ROP2-P30复合重组蛋白疫苗免疫BALB/c小鼠诱导机体产生大量的IgM、IgG抗体和IFN-γ、IL-2及IL-4细胞因子。攻击试验表明,复合重组蛋白免疫组和对照组相比,小鼠的存活时间明显延长。结论ROP2-P30复合重组蛋白免疫BALB/c小鼠诱导其产生高水平的体液和细胞免疫应答,该复合重组蛋白是抗刚地弓形虫感染的候选疫苗之一。     

弓形虫PRU株慢性感染小鼠脑部包囊的时空分布特征

目的观察实验小鼠脑内包囊的时空分布特点,为探讨弓形虫感染引起的情志和行为改变提供病理基础。方法弓形虫PRU株经口感染小鼠,经HE染色和免疫组化检测,在显微镜下计算前额、海马、丘脑、小脑和杏仁核部位的包囊个数,然后随机选取上述部位的5个视野拍照,计算包囊的平均密度,并用方差分析比较不同脑组织包囊密度有无统计学差异。结果弓形虫感染30和90d时,HE染色和免疫组化后显微镜观察发现,小鼠不同位置弓形虫包囊密度差异有统计学意义,其中,丘脑的包囊密度最大,其次是前额皮质、海马、杏仁核,小脑的包囊密度最小。丘脑的包囊密度显著高于其他4个脑区（P〈0.01）,小脑的包囊密度显著低于其它4个部位（P〈0.01）,而前额皮质、海马和杏仁核所含的包囊密度差异无统计学意义（P〉0.05）;弓形虫感染1个月时,杏仁核包囊密度明显低于感染3个月（F=18.314,P〈0.001）,但是小脑的包囊密度高于感染3个月（F=18.314,P〈0.001）。结论弓形虫包囊在慢性感染小鼠不同脑组织内的分布具有时空特异性,这可能是其临床表现的病理学基础。