我国不同流行区婴儿利什曼原虫前鞭毛体比较蛋白质组学分析

目的 应用比较蛋白质组学方法分析两株来自我国内脏利什曼病不同流行区的婴儿利什曼原虫前鞭毛体蛋白表达差异,为筛选和鉴定我国不同流行区利什曼原虫致病差异相关分子奠定基础。方法 分别培养两株分离于我国四川九寨沟县（SC6株）和新疆伽师县内脏利什曼病流行区（JIASHI?5株）的婴儿利什曼原虫前鞭毛体,经酶解后进行液相色谱串联质谱（Liquid chromatography?tandem mass spectrometry, LC?MS/MS）非标记（Label?free）定量分析,利用MaxQuant软件（版本号1.3.0.5）查库,进行非标定量（Label?free quantitation,LFQ）分析。结果 成功鉴定蛋白4 274个,筛选出差异蛋白1 219个（差异倍数＞2.0 或 ＜0.5,P < 0.05）,其中JIASHI?5株前鞭毛体特有蛋白550个,SC6 株前鞭毛体特有蛋白174个。SC6 株和 JIASHI?5 株间差异表达蛋白495个,其中SC6株上调蛋白（高表达）167个,JIASHI?5株上调蛋白328个。这些差异表达蛋白主要涉及能量代谢、应激反应、延长感染宿主细胞的寿命以及利什曼原虫存活与增殖。结论　来自我国内脏利什曼病不同流行区的婴儿利什曼原虫前鞭毛体蛋白质表达存在差异。     

狂犬病两株不同毒力克隆株病毒的神经毒力和全长基因序列比较

目的 对2株毒力不同的狂犬病病毒克隆株(CTN181-3和CTN181-12)进行神经毒力和全长基因序列的比较。方法 分别对2株病毒用10、14和21日龄小鼠脑内接种,测定小鼠的致死力(LD₅₀);同时用空斑法测定2株病毒的病毒滴度(PFU),以lgPFU/lgLD₅₀比较2株病毒的毒力差异;分别对2株病毒进行全基因序列测定并分析,找出毒力差异关键基因位点。结果 2株病毒对10、14和21日龄小鼠的lgPFU/lgLD₅₀,CTN181-3株为0.07、3.40和6.60,CTN181-12株为<-0.9、<-0.6和1.04。全基因序列分析表明,二者共有8个核苷酸和6个位点的氨基酸存在差异。结论 CTN181-3株的神经毒力明显低于CTN181-12株,两者间6个氨基酸的差异是其毒力差异的分子基础。     

不同种（株）利什曼原虫毒力相关基因的表达差异

目的 观察不同种（株）利什曼原虫前鞭毛体和无鞭毛体的毒力相关基因表达情况。 方法 制备杜氏利什曼原虫、婴儿利什曼原虫、热带利什曼原虫、硕大利什曼原虫和墨西哥利什曼原虫等5种7株利什曼原虫前鞭毛体和无鞭毛体的总RNA,采用半定量RT-PCR法,以α-微管蛋白基因和3-磷酸甘油醛脱氢酶基因（GAPDH）作为阳性对照,根据GenBank公布的GDP甘露糖焦磷酸酶基因（GDPMP）、A2抗原相关蛋白基因（A2rel）、脂磷酸多糖合成蛋白1基因（LPG1）、脂磷酸多糖合成蛋白2基因（LPG2）、动基体膜蛋白11基因（KMP-11）、胱氨酸蛋白酶C基因（CPC）、亲水性酰化表面蛋白B1基因（HASPB1）、胱氨酸蛋白酶2基因（CPB2）、胱氨酸蛋白酶B2.8基因（CPB2.8）和热激蛋白100基因（CLP b）等毒力相关基因的核苷酸序列,设计特异性引物进行RT-PCR扩增,分析以上各基因在各种（株）前鞭毛体和无鞭毛体中的表达情况。 结果 各毒力基因在不同种（株）利什曼原虫的前鞭毛体和无鞭毛体中的表达明显不同,HASPB1基因在7个种（株）利什曼原虫的无鞭毛体和杜氏利什曼原虫前鞭毛体中均表达,GDPMP、LPG1、LPG2、CPB2.8、CPB2、A2rel和CLP基因分别在特定种（株）的前鞭毛体和/或无鞭毛体中表达,CPC基因仅在杜氏利什曼原虫SC10株和硕大利什曼原虫无鞭毛体内表达,KMP-11基因在7个种（株）利什曼原虫前鞭毛体或无鞭毛体内均不表达。 结论 毒力相关基因的表达存在种特异性和期特异性。     

杜氏利什曼原虫无鞭毛体表达抗原的Western blot和MALDI-TOF/TOF分析

目的鉴定杜氏利什曼原虫无鞭毛体特异表达抗原。方法培养杜氏利什曼原虫前鞭毛体并体外转化无鞭毛体,其总蛋白经2-DE电泳后以小鼠抗杜氏利什曼原虫无鞭毛体血清进行Western blot,对前鞭毛体与无鞭毛体特异表达抗原蛋白进行MALDI-TOF/TOF串联质谱鉴定。重组表达无鞭毛体特异表达抗原编码基因,以Western blot法对重组蛋白进行鉴定。结果等量的杜氏利什曼原虫前鞭毛体与无鞭毛体蛋白经2-DE电泳均可呈现680~742个蛋白点,Western blot及MALDI-TOF/TOF-MS分析甘油醛3-磷酸脱氢酶与延伸因子2为杜氏利什曼原虫前鞭毛体特异表达抗原,核苷二磷酸激酶为无鞭毛体特异表达抗原。重组核苷二磷酸激酶编码基因表达产物经Western blot证实为杜氏利什曼原虫无鞭毛体特异表达强抗原。结论杜氏利什曼原虫前鞭毛体与无鞭毛体抗原表达存在差异,核苷二磷酸激酶为杜氏利什曼原虫无鞭毛体特异表达强抗原。     

不同种株利什曼原虫的比较蛋白质组学分析

目的观察不同种株利什曼原虫前鞭毛体蛋白质表达状况。方法制备杜氏利什曼原虫四川SC6株、杜氏利什曼原虫四川SC10株和硕大利什曼原虫5ASKH株前鞭毛体总蛋白,以pH范围3-10的预制胶条进行双向电泳(2-D),考马斯亮蓝染色,PDQust软件分析凝胶,主要差异蛋白点用电喷雾质谱法进行鉴定。结果等量的杜氏利什曼原虫四川SC6株、杜氏利什曼原虫四川SC10株和硕大利什曼原虫5ASKH株前鞭毛体总蛋白均获近700个蛋白点,不同种株利什曼原虫前鞭毛体蛋白质2-D图谱中,14蛋白点呈恒定差异表达,从中鉴定出10个功能明确的蛋白质,分别具有下列生物功能:糖代谢与磷脂合成(烯醇酶、变旋酶、NADP依耐乙醇脱氢酶、乙醇胺磷酸胞苷酸转移酶),压力反应(细胞内过氧化物酶、锥虫还原蛋白过氧化物酶),细胞膜/细胞骨架(α-微管蛋白、β-微管蛋白),核酸代谢(琥珀酰辅酶A连接酶(GDP形成)、内源性RNA酶L-PSP(pb5)),细胞周期与增殖(延伸因子2)。结论不同种株利什曼原虫前鞭毛体蛋白质的表达存在不同,为理解不同种株利什曼原虫的毒力、免疫原性和代谢特征提供了新的视角。     

鼠疫耶尔森菌小肽基因sORF34缺失株毒力及短期免疫保护效果评价

目的 探索小肽基因34(sORF34)缺失对鼠疫耶尔森菌毒力影响,并评价sORF34缺失株免疫保护活性。方法 基于鼠疫耶尔森菌201株和鼠疫耶尔森菌EV76疫苗株利用λ-Red一步法构建小肽基因缺失株。比较鼠疫小肽基因缺失株和亲本株之间的毒力差别,并采用皮下免疫方式对小鼠进行免疫,与EV76疫苗株比较,评价体液免疫、保护率等方面的差异。结果 PCR扩增结果证实,小肽基因缺失株构建成功。通过皮下LD₅₀测定、生存曲线测定,表明小肽基因缺失株较亲本株毒力下降。鼠疫201ΔsORF34和EV76ΔsORF34皮下免疫后可刺激机体产生抗F1-IgG,其中鼠疫201ΔsORF34免疫组抗体滴度与EV76疫苗株免疫组差异无统计学意义(P>0.05),EV76ΔsORF34免疫组较EV76疫苗株免疫组抗体滴度低;鼠疫EV76ΔsORF34株可刺激机体产生低滴度抗LcrV-IgG,而EV76和201ΔsORF34株不能刺激机体产生抗LcrV-IgG。初免后42 d使用致死剂量鼠疫201株进行皮下攻毒和滴鼻攻毒,3组保护率差异无统计学意义(P>0.05)。结论 小肽基因缺失株经皮下途径感染BALB/c小鼠的毒力下降,鼠疫EV76ΔsORF34株较EV76疫苗株残存毒力进一步下降,但保护率差异无统计学意义(P>0.05),EV76ΔsORF34株有作为鼠疫减毒活疫苗候选株的潜力。     

杜氏利什曼原虫前鞭毛体和无鞭毛体的比较蛋白质组学分析

目的 应用蛋白质组学技术分析杜氏利什曼原虫前鞭毛体和无鞭毛体蛋白质表达状况。 方法 分别提取和纯化杜氏利什曼原虫四川SC6株前鞭毛体与纯培养无鞭毛体的总蛋白,分别经pH3～10的预制胶条进行双向电泳分离,凝胶用考马斯亮蓝染色,凝胶图像以PDQuest 1.0软件分析,并对主要差异表达蛋白点用电喷雾质谱法进行鉴定。 结果 等量的前鞭毛体与纯培养的无鞭毛体总蛋白经双向电泳分离后均可获近700个蛋白点,其中超过90%的蛋白点的分布和相对强度基本一致。与前鞭毛体比较,6个蛋白点在无鞭毛体蛋白中明显高表达,3个蛋白点低表达。6个明显高表达的蛋白点中有5个为已知功能蛋白,分别为Reiske铁硫蛋白前体、α微管蛋白、过氧化物酶1、二氢硫辛酰胺乙酰转移酶前体和甘露糖-1-磷酸瓜氨酸转移酶;3个低表达的蛋白点中有2个为已知功能蛋白,分别为热休克蛋白70和β微管蛋白。这些差异调节表达蛋白与碳水化合物/能量代谢,应激反应,细胞膜和细胞骨架形成相关。 结论 前鞭毛体与无鞭毛体蛋白质的表达存在差异。     

杜氏利什曼原虫四川分离株无鞭毛体蛋白基因的克隆及真核表达

目的克隆、真核表达杜氏利什曼原虫四川株的无鞭毛体蛋白(amastin)编码基因。方法PCR扩增杜氏利什曼原虫四川汶川人株L．d．SC10H2与四川南坪犬株L．d．SC7的无鞭毛体蛋白基因,将该基因导入pcDNA3．1( ),构建真核表达重组质牲,转染NIH3T3细胞,采用免疫荧光法鉴定重组质粒的瞬时表达;RT-PCR和Western blotting鉴定稳定表达,结果2株杜氏利什曼原虫均扩增出552 bp的无鞭毛体蛋白基因。同源性为86％。转染后在NIH3T3细胞膜和细胞内观察到较强的绿色荧光,表明无鞭毛体蛋白基因在NIH3T3细胞中获得短暂表达。细胞裂解产物经Western blotting,在相对分子质量(Mr)约20000处检测到阳性杂交信号,表明无鞭毛体蛋白基因在NIH3T3细胞内获得了稳定表达。结论获得了我国杜氏利什曼原虫四川分离株L．d．SC10H2和L．d．SC7的无鞭毛体蛋白基因序列,并在NIH3T3细胞中稳定表达。     

墨西哥利什曼原虫无鞭毛体蛋囟的基因克隆化与序列分析

目的　克隆墨西哥利什曼原虫(L.mer)WR972株的无鞭毛体蛋白(amastin)的编码基因,并对其同源核苷酸序列进行分析。方法　根据已克隆的亚马逊利什曼原虫无鞭毛体蛋白的编码基因序列,设计并合成无鞭毛体蛋白基因特异性引物,以墨西哥利什曼原虫VR972株的基因组DNA作为模板,进行多聚酶链反应(PCR)扩增。将扩增的DNA片段克隆到pCR2.1T载体中,进行测序,并对同源的核苷酸序列分析、比较。结果　从体外培养的墨西哥利什曼原虫WR972株提取基因组DNA,以无鞭毛体蛋白基因特异性引物进行PCR扩增获得550bp的DNA片段。克隆到pCR2.1T载体片段进行的序列测定结果　表明,获得了墨西哥利什曼原虫的无鞭毛体蛋白编码基因片段,与亚马逊利什曼原虫的无鞭毛体蛋白基因之间具有高度的同源性。结论　实现了墨西哥利什曼原虫无鞭毛体蛋白基因的克隆化,为进一步研究其表达及作为疫苗研究的候选基因奠定了基础。     

陕西省韩城市利什曼原虫SSU rRNA基因系统发育研究

目的 了解陕西省韩城市黑热病区利什曼原虫种类及分子遗传特征。方法 对来自韩城市的利什曼原虫阳性标本,进行SSU rRNA基因序列扩增,扩增产物进行测序,与其它不同种类、不同地区利什曼原虫的SSU rRNA基因序列进行比对,分析突变位点规律,建立系统发育树。结果 有3份标本SSU rRNA基因序列扩增测序成功,其基因序列与杜氏利什曼原虫、夏科氏利什曼原虫、婴儿利什曼原虫的SSU rRNA序列同源性较高,与婴儿利什曼原虫在UQ-II突变区有一个位点的变异,并与新疆荒漠虫株、甘肃省利什曼虫株聚为一类。结论 结合流行病学与SSU rRNA序列分析结果,陕西省韩城市黑热病病原体应为婴儿利什曼原虫。     

Development of infective stage Leishmania promastigotes within phlebotomine sand flies

Midgut promastigotes were obtained from Phlebotomus papatasi and Lutzomyia longipalpis on days 3-7 after infection with cloned isolates of Leishmania major and Leishmania mexicana amazonensis, respectively, and examined as to their ability to initiate cutaneous infections in BALB/c mice. Sequential development of midgut promastigotes from a noninfective to an infective stage was confirmed for both the New World and Old World species. The generation of infective promastigotes from rapidly dividing avirulent populations occurred as early as day 3 and was well under way by day 4 after infective feed. Optimally infective promastigotes were recovered from midguts shortly after bloodmeal passage, coinciding with the time at which another bloodmeal is sought by the fly.     

Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum

The obligate intracellular pathogen Leishmania major survives and multiplies in professional phagocytes. The evasion strategy to circumvent killing by host phagocytes and establish a productive infection is poorly understood. Here we report that the virulent inoculum of Leishmania promastigotes contains a high ratio of annexin A5-binding apoptotic parasites. This subpopulation of parasites is characterized by a round body shape, a swollen kinetoplast, nuclear condensation, and a lack of multiplication and represents dying or already dead parasites. After depleting the apoptotic parasites from a virulent population, Leishmania do not survive in phagocytes in vitro and lose their disease-inducing ability in vivo. TGF-beta induced by apoptotic parasites is likely to mediate the silencing of phagocytes and lead to survival of infectious Leishmania populations. The data demonstrate that apoptotic promastigotes, in an altruistic way, enable the intracellular survival of the viable parasites.     

Destruction of Leishmania mexicana amazonensis promastigotes by normal human serum

Fresh normal human serum was observed to have a lethal effect on Leishmania mexicana amazonensis promastigotes obtained from laboratory-bred Lutzomyia longipalpis or on promastigotes grown in liquid culture medium, inoculated with the same isolates. Heat inactivation abolished the Leishmania lytic activity from the sera. Resistance of culture promastigotes to lysis by normal human serum was investigated in three isolates of L. m. amazonensis. Development of resistance (up to 7%) was found in only one isolate, obtained from the bone marrow in a human case of visceral leishmaniasis.     

Effects of long-term in vitro cultivation on the virulence of cloned lines of Leishmania major promastigotes.

The virulences of several clones from a single Leishmania major strain were studied in BALB/c mice. Clones showed the same pattern of infectivity and virulence two months after cloning as the parental population. After prolonged in vitro culture, however, it was apparent that two types of virulent clones existed: although the level of virulence remained stable in some clones, in others, such as C-11, it progressively decreased, as in the parental population. The progressive loss in virulence in a continuously cultured mixed population was probably due to selection, as the initial mixture of a stable virulent clone and a stable avirulent clone eventually yielded a totally avirulent promastigote population. After cultivation for 12 months, neither clone C-11 nor the parental population produced lesions in inoculated mice but virulent parasites were recovered from the inguinal nodes of the mice, possibly as a result of selection in vivo for virulent parasites.     

Infectivity and attenuation of Leishmania donovani promastigotes. II: Association of the loss of parasite infectivity with the terminal galactosylation of precursor acceptors present in virulent parasites by the developmentally regulated galactosyltransferase

The beta1-4 galactosyltransferase enzyme of the Leishmania donovani promastigotes, was found to be developmentally regulated and expressed only in the attenuated parasites. The enzymatic product of soluble determinants of virulent promastigotes and the galactosyltransferase enzyme was found to stimulate the macrophage burst activity but inhibit in vitro intracellular parasitism. In contrast, removal of terminal galactose moieties from soluble determinants of attenuated parasites resulted in the inhibition of macrophage respiratory burst activity but did not now inhibit intracellular parasitism. We propose that the terminal galactosylation of acceptor substrates present in virulent parasites by the developmentally regulated galactosyltransferase is associated with loss of parasite virulence.     

Leishmania major: resistance of promastigotes to paromomycin, and susceptibility of amastigotes to paromomycin-methylbenzethonium chloride ointment.

Cutaneous lesions caused by Leishmania major in BALB/c mice were cured completely when treated topically with an ointment comprising 15% paromomycin sulphate and 1-2% methylbenzethonium chloride ointment in soft white paraffin twice daily for 10 days. No parasites were detected in tissue smears or in cultures from treated cutaneous lesions. Re-developing lesions, considered to be resulting from the migration of parasites from internal organs, showed almost the same response to topical treatment. Promastigotes of the virulent clone 121 of L. major LRC-L137 which were exposed to 100 micrograms ml-1 of paromomycin in RPMI medium at 28 degrees C developed resistance to the drug over 10 passages of exposure. Enzyme analysis of susceptible and resistant promastigotes of this clone showed no differences with regard to their profiles based on 11 enzymes.