Transient transfection of the enteric parasite Entamoeba histolytica and expression of firefly luciferase.

Development of DNA-mediated transfection in Entamoeba histolytica will facilitate basic research toward the control of this protozoan parasite. A transient transfection system was established by using the firefly luciferase gene ligated to the 5' and 3' flanking regions of the amebic hgl1 gene. The optimal construct tested encoded an hgl1-luciferase fusion protein and contained 1 kb of 5' flanking sequence with 16 bases of coding sequence from the hgl1 gene ligated in-frame to the luciferase start codon and 2.3 kb of 3' flanking sequence from hgl1 ligated 3' to the luciferase stop codon. Optimal electroporation conditions in strain HM-1:IMSS trophozoites when using this construct were 500 microF and 500 V/cm, which resulted in luciferase activity up to 5000-fold above background 9-12 hr after electroporation. Constructs that contained the luciferase gene without amebic flanking sequences or that contained a simian virus 40 promoter, enhancer, and polyadenylylation signal produced only background levels of luciferase activity. The ability to introduce and express genes in amebae will now permit a genetic analysis of the virulence of this organism, which remains a serious threat to world health.     

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.     

Transfection of the malaria parasite and expression of firefly luciferase.

The goal of this work is to develop a method for the functional analysis of malaria genes using the method of DNA transfection. We have developed a transient transfection vector by constructing a chimeric gene in which the firefly luciferase gene was inserted in frame into the coding region of the pgs28 gene of Plasmodium gallinaceum. This plasmid DNA was introduced into P. gallinaceum gametes and fertilized zygotes by electroporation, and luciferase expression was assayed after 24 hr. This report of successful introduction and expression of a foreign gene in a malaria parasite demonstrates the feasibility of this approach to developing methods for the functional analysis of parasite genes.     

阳离子脂质体介导的虫荧光素酶基因表达

目的 研究阳离子脂质体Lipofectin介导虫荧光素酶基因在不同细胞株中的表达。方法 质粒pDR2luc在大肠杆菌DH5a中扩增后用碱裂解法提取,并经Sepharose 2B凝胶过滤柱层析,通过凝胶电泳及紫外分光光度计分析纯度、定量,以Lipofectin分别转染人肝癌细胞株HepG2、SMMC7721、人肾癌细胞株GRC及非洲绿猴肾细胞COS7,然后以液体闪烁计数仪单光子计数法测定荧光素酶活性。结果 荧光素酶活性在4种细胞株中均有表达,且均有显著性差异(均P<0.05)。结论 阳离子脂质体Lipofectin可用于多种真核细胞基因转染。     

蓝氏贾第鞭毛虫胞外核酸酶的表达纯化和活性鉴定

目的 克隆、原核表达蓝氏贾第鞭毛虫(Giardia lamblia,贾第虫)的胞外核酸酶编码区,并对其蛋白产物进行活性鉴定。方法 对贾第虫胞外核酸酶(GeNuc)蛋白进行生物信息学分析,根据分析结果以C2株贾第虫基因组DNA为模板扩增获得GeNuc去信号肽段编码区序列,双酶切连入原核表达载体pET-28a(+),将酶切和测序验证正确的重组质粒转化E.coli Rosetta(DE3),经IPTG诱导表达融合蛋白,SDS-PAGE及Western blot鉴定蛋白产物。Ni-NTA亲和层析纯化GeNuc蛋白,经复性后验证其对质粒DNA的水解能力。结果 成功克隆了长约800 bp的GeNuc编码区并构建了原核表达载体pET-28a(+)-GeNuc,测序结果显示C2株GeNuc序列与WB株相同;在大肠杆菌中诱导表达获得了相对分子量约30.8 kDa的融合蛋白;复性后的纯化GeNuc蛋白具有降解双链DNA的能力,但活性较商品化DNaseⅠ低。结论 证明了GeNuc的存在,为GeNuc抗体的制备及贾第虫致病机制的研究提供了实验材料。     

蓝氏贾第鞭毛虫基因表达调控的研究进展

蓝氏贾第鞭毛虫是一种重要的致病性寄生虫,人体感染后主要引起腹泻和营养不良等症状。作为一种源真核生物,其基因表达调控机制可能与其他真核生物有较大的区别。本文从蓝氏贾第虫鞭毛虫启动子、转录因子、转录后调节、翻译起始和表观遗传学等方面,对贾第虫基因表达调控的研究进展进行综述。     

蓝氏贾第鞭毛虫末端结合蛋白 1 基因的克隆与原核表达

目的 克隆并原核表达 C2 株蓝氏贾第鞭毛虫(Giardia lambia,简称贾第虫)末端结合蛋白 1(End-bind- ing protein 1,geb1)基因,获得重组 gEB1 蛋白。 方法 由于 geb1 基因无内含子,我们以 C2 株贾第虫基因组为模板,以国际标准株WB 株geb1 基因序列为参考序列,设计引物克隆 geb1 基因,经 NcoⅠ和 XhoⅠ双酶切与原核表达载体 pET-28α(+)连接,转化感受态 E. coli TOP10,经筛选和测序验证后导入大肠杆菌 E. coli Rosetta(DE3),异丙基-β-D -硫代半乳糖(IPTG)诱导 gEB1 蛋白表达,产物经 SDS-PAGE 和 Western blot 进行检测和验证。 结果 成功构建了表 达 C2 株贾第虫 geb1 基因的原核表达载体 pET-28α(+)- gEB1,转化入大肠杆菌 E. coli Rosetta(DE3),重组菌株经 0. 1 mmol / L IPTG ,30℃低温诱导 5 h, SDS-PAGE 和 Western blot 显示,在相对分子量约 29 KDa 的位置出现目的蛋 白条带,与理论值一致。 结论 用大肠杆菌成功表达了 gEB1 蛋白,为 gEB1 蛋白的功能研究和抗体制备提供了材料。     

蓝氏贾第虫病毒体外转录体转染条件的研究

目的 确定蓝氏贾第虫病毒体外转录体电穿孔转染的最佳条件。方法 以不同的电击缓冲液、电压及脉冲时间应用GLV和GFP嵌合体体外转录体电穿孔转染蓝氏贾第虫，测定转染虫体的存活率及GFP表达量。结果 在不同转染条件下GLV和GFP嵌合体体外转录体均能成功转染蓝氏贾第虫，且在cytomix缓冲液、1000V／cm、8ms电击条件下，对虫体损伤最小，GFP表达量最高。结论 本试验确定的蓝氏贾第虫病毒体外转录体电穿孔转染最佳条件是电击缓冲液为cytornix缓冲液，电压为1000V／cm、脉)中时间为8ms。     

Giardia lamblia as an intestinal pathogen.

Giardia lamblia are protozoan parasites which cause human intestinal disease. The life cycle has a multiplying intraduodenal trophozoite and an excreted cyst. Infection occurs after cyst ingestion from faecally contaminated water or by direct faecal-oral transmission in situations of poor sanitary standards, but the zoonotic nature of giardiasis is debated. The pathophysiology may arise from enzyme or active transport deficiencies, synergy with intestinal bacteria or an immunopathological process. Diagnosis is made by microscopic identification of cysts or trophozoites in small bowel samples or faeces. Symptoms are acute with diarrhoea (without blood), abdominal cramps, bloating and flatulence. The treatment of choice is either metronidazole or tinidazole. No vaccine or drug prophylaxis exists, and measures to avoid cyst ingestion should be undertaken.     

蓝氏贾第鞭毛虫的细胞骨架蛋白

蓝氏贾第鞭毛虫是一种重要的肠道致病性原虫,其致病力与细胞骨架密切相关。贾第虫具有高度发达的细胞骨架系统,包括8根鞭毛、1个腹吸盘、中体和funis等,主要由微管蛋白、肌动蛋白、动力蛋白、贾第素及其相关蛋白组成。该文就贾第虫细胞骨架及相关蛋白的结构和功能作一综述。     

A spliceosomal intron in Giardia lamblia

Short introns occur in numerous protist lineages, but there are no reports of intervening sequences in the protists Giardia lamblia and Trichomonas vaginalis, which may represent the deepest known branches in the eukaryotic line of descent. We have discovered a 35-bp spliceosomal intron in a gene encoding a putative [2Fe-2S] ferredoxin of G. lamblia. The Giardia intron contains a canonical splice site at its 3' end (AG), a noncanonical splice site at its 5' end (CT), and a branch point sequence that fits the yeast consensus sequence of TACTAAC except for the first nucleotide (AACTAAC). We have also identified several G. lamblia genes with spliceosomal peptides, including homologues of eukaryote-specific spliceosomal peptides (Prp8 and Prp11), several DExH-box RNA-helicases that have homologues in eubacteria, but serve essential functions in the splicing of introns in eukaryotes, and 11 predicted archaebacteria-like Sm and like-Sm core peptides, which coat small nuclear RNAs. Phylogenetic analyses show the Giardia Sm core peptides are the products of multiple, ancestral gene duplications followed by divergence, but they retain strong similarity to Sm and like-Sm peptides of other eukaryotes. Although we have documented only a single intron in Giardia, it likely has other introns and fully functional, spliceosomal machinery. If introns were added during eukaryotic evolution (the introns-late hypothesis), then these results push back the date of this event before the branching of G. lamblia.     

Isolation and expression of the gene for a major surface protein of Giardia lamblia.

To study the interactions between the parasitic protozoan Giardia lamblia and its environment, we have cloned the gene that encodes the two major surface-labeled trophozoite protein species. Sequence analysis of this gene reveals a single open reading frame specifying a hydrophilic, cysteine-rich (11.8%) protein of 72.5-kDa molecular mass with an amino-terminal signal peptide and a postulated hydrophobic membrane-spanning anchor region near the carboxyl terminus. Most of the cysteine residues (58 of 84) are in the motif Cys-Xaa-Xaa-Cys, which is dispersed 29 times throughout the sequence. Antibodies against the recombinant protein react with the entire surface of live trophozoites, including flagella and adhesive disc. These antibodies inhibit trophozoite attachment, prevent growth, and immunoprecipitate the major approximately 66- and 85-kDa proteins from surface-labeled live trophozoites. The recombinant Escherichia coli also expresses polypeptides of approximately 66- and 85-kDa molecular mass, which are not fusion proteins. This suggests that the processing and/or conformational changes that lead to production of these two peptide species in E. coli reflect those that occur in Giardia. The abundance of cysteine residues suggests that the native proteins on the parasite surface may contain numerous disulfide bonds, which would promote resistance to intestinal fluid proteases and to the detergent activity of bile salts and would help to explain the survival of Giardia in the human small intestine.     

Association between Whipple's disease and Giardia lamblia infection.

Whipple' disease is mainly characterized by affecting the digestive system, although it can be a multisystemic process with different clinical symptoms. The bacillus causing the disease has been isolated and cultivated in 2000 and the genome sequence has been recently analyzed in 2003, which means new perspectives for its diagnosis and treatment. Giardiasis is an infestation caused by a protozoo and may cause a malabsorption syndrome or run in a subclinic way. The case of a middle-aged male is described, who after a three-year period of migratory arthralgias, showed weight loss, diarrheas and abdominal pain, being diagnosed of Giardiasis, and after the persistent symptoms and a number of studies, was diagnosed with Whipple disease. Nineteen cases of Giardia-Whipple coinfection have been described in the literature, but the reason of this association has not been found yet. The discussion on whether there is an alteration in the immunitary system which facilitates infections or, the development of an infection lead to the other one, goes on.     

Giardia lamblia: phospholipid analysis of human isolates.

Thin layer chromatograms for phospholipids obtained from 11 human Giardia lamblia isolates and their culture media have shown that phosphatidylcholine and sphingomyelin are the predominant phospholipid classes in all samples. A decrease in the relative percentage of the different classes, especially of phosphatidylcholine, was noticed in the medium after Giardia growth. Fatty acid analysis of the parasite phosphatidylcholine demonstrated that while oleate and palmitate were the major fatty acids in most isolates, arachidonate predominated in two of those studied. Some isolates contained small amounts of myristate, which was not present in the phosphatidylcholine of the culture medium. Moreover, stearate and linoleate predominated in phosphatidylcholine obtained from both media types. The saturated/unsaturated fatty acid ratio also varied for the different isolates. These results appear to suggest heterogeneity in the metabolic activity and utilization of lipid molecules between Giardia isolates.     

蓝氏贾第鞭毛虫遗传多样性研究进展

蓝氏贾第鞭毛虫是全球危害人类健康的重要寄生性原虫之一。由于该虫宿主包括几乎所有脊椎动物,虫种的基因型和表现型变异较大,因而其宿主特异性及人畜共患传播问题尚未完全解决。近年来,贾第鞭毛虫病的广泛流行已引起人们对其遗传多样性的关注。基于PCR的分子鉴定方法及多种分子标志的应用使得对贾第鞭毛虫种群的遗传结构有了更多了解。该文就蓝氏贾第鞭毛虫遗传多样性的研究进展作一综述。     

蓝氏贾第鞭毛虫感染的免疫学诊断方法

蓝氏贾第鞭毛虫感染可引起人及其他哺乳动物腹泻.近年来,贾第虫病的严重性和危害性日益受到重视.本文对蓝氏贾第鞭毛虫感染的免疫学诊断方法研究进行了综述,比较了各种方法的优缺点,简要介绍了一些商品化试剂盒的使用情况.贾第虫病免疫学诊断方法的应用,对贾第虫病的临床诊断、治疗、预后将带来极大的方便.     

蓝氏贾第鞭毛虫的免疫逃避机制

蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)是一种世界性分布的机会性致病原虫,能够引起以腹泻为主要表现的贾第虫病,严重影响人类尤其是儿童的健康和发育。在贾第虫感染过程中,宿主的非特异性和特异性免疫系统均会产生强烈的抗贾第虫效应,但贾第虫通过抗原漂变、L-精氨酸饥饿等机制逃避和抑制宿主的免疫反应,引起宿主的长期或反复感染。本文对宿主的抗贾第虫免疫以及贾第虫的免疫逃避机制做一综述。