柔嫩艾美耳球虫新基因的生物信息学分析与克隆表达

为了研究柔嫩艾美耳球虫(Eimeria tenella)的两个主要入侵发育阶段——子孢子和裂殖子的差异基因,根据已报道的子孢子与裂殖子差异的ESTs序列,应用RACE技术克隆获得了子孢子阶段差异表达的新基因全长cDNA序列,命名为ZL583.该基因全长为862 bp,开放阅读框(ORF)为486 bp,编码161个氨基酸,编码蛋白的分子量约为16.9 kD,利用生物信息学分析软件,分析新基因所编码蛋白的性质、定位、结构等特征.利用荧光定量PCR对柔嫩艾美耳球虫不同发育阶段该基因的表达量进行分析显示,子孢子阶段的表达高于其他发育阶段.将ZL583克隆于pET-28a中构建重组质粒,转化大肠杆菌BL21(DE3)后经IPTG诱导表达,获得重组蛋白分子量约23 kD.免疫印迹分析显示该重组蛋白可与兔抗子孢子血清发生特异性反应,表明该蛋白具有较好的反应原性.该结果为进一步研究该基因的生物学功能奠定基础.     

柔嫩艾美耳球虫基因在丝状体蓝藻中的克隆

以纯化的柔嫩艾美耳球虫的孢子化卵巢为模板,根据已知的序列设计一对引物,用RT－PCR方法扩增出球虫的S07基因,通过三亲接合转移、新霉素筛选、序列分析等方法得到了转球虫基因工程蓝藻。这为球虫基因在蓝藻中表达奠定了基础。     

柔嫩艾美耳球虫配子发生的超微结构

利用透射电镜对柔嫩艾美耳球虫配子生殖阶段的超微结构进行了,大配子体和小配子体于相邻的宿主肠上皮细胞内相产生,由末代裂殖子入后,长大变圆而形成,小配子的形成为直接分化型,首先细胞核分裂成为多核体,随后细胞核向周边移动,然后紧靠细胞处的限制向外突出,临近突出部位的限制膜下陷,在核上方形成中心粒,中心粒发育为基粒,鞭毛中的微管和附着微管,早期形成的小配子仍与小配子体的殖体相连,成熟的小配子与配子体分离,外型香蕉状,外被单位膜,内有一电子结构十分致密的细胞核,核的头端侧面有一个巨大的线粒体,小配子有鞭毛2根,每根鞭毛内有微管,组成为9＋2结构,此外,小配子至少有6根附着微管,大配子体和大配子外被单位膜,内部形成大量的成囊体1和成囊体2,并有大量的支链淀粉和脂肪体,中央有一个细胞核,卵囊臂有5层,细胞核位于细胞中央,细胞内有大量的支链淀粉和脂肪体。     

柔嫩艾美耳球虫广东株子孢子折光体抗原Etp28基因的克隆和序列分析

鸡球虫病是由顶器官亚门（Ａｐｉｃｏｍｐｌｅｘａ）艾美耳属（Ｅｉｍｅｒｉａ）的一种单细胞寄生原虫引起的严重危害家禽生长发育的疾病。其中,柔嫩艾美耳球虫（Ｅｉｍｅｒｉａｔｅｎｅｌａ）通过在鸡的盲肠上皮细胞内进行裂体生殖,形成大量的裂殖体并引起广泛出血而造...     

柔嫩艾美耳球虫感染对鸡盲肠中益生菌的影响

双歧杆菌（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍｓｐｐ．）及乳酸杆菌（Ｌａｃｔｏｂａｃｉｌｌｕｓｓｐｐ．）被认为是动物机体内重要的有益微生物菌群之一。本文对鸡感染柔嫩艾美耳球虫后盲肠中上述两种微生物的变化情况进行了研究。结果表明：鸡在感染球虫后第４、７、１０ｄ时盲肠中双歧杆菌的数量显著减少（Ｈ＜０．０５）;在球虫感染后的第４、７、１４ｄ时,盲肠中的乳酸杆菌呈显著下降趋势（Ｐ＜０．０５）。     

柔嫩艾美耳球虫基因在丝状体蓝藻中的克隆*

以纯化的柔嫩艾美耳球虫的孢子化卵囊为模板 ,根据已知的序列设计一对引物 ,用RT PCR方法扩增出球虫的SO7基因 ,通过三亲接合转移、新霉素筛选、序列分析等方法得到了转球虫基因工程蓝藻。这为球虫基因在蓝藻中表达奠定了基础。     

柔嫩艾美耳球虫钙依赖蛋白激酶3基因在毕赤酵母中的表达及分析

旨在真核表达系统中高效表达柔嫩艾美耳球虫钙依赖蛋白激酶3(Eimeria tenella calcium-dependent protein kinase-3,EtCDPK3),获得有活性的天然蛋白,利用毕赤酵母表达系统对该基因进行了表达.将EtCDPK3基因连接到毕赤酵母表达载体pPIC9K上,构建重组质粒pPIC9K-EtCDPK3.重组质粒通过电击转化入酵母细胞GS115后,用组氨酸缺陷培养基和G418分别进行筛选,获得含重组质粒的酵母表达细胞.重组酵母细胞在含1％甲醇的BMMY培养基中诱导产生目的蛋白,培养收集1-4d的部分上清.经SDS-PAGE检测,所表达的蛋白相对分子质量约为49 kD.Western blotting表明,该蛋白能与兔抗EtCDPK3血清特异性结合.结果表明,柔嫩艾美耳球虫CDPK3基因在毕赤酵母中成功地进行了表达.     

柔嫩艾美球虫体外脱囊试验和观察

用机械方法破碎柔嫩艾美球虫卵囊后,其孢子囊可在体外接受含0.3％胰蛋白酶和5.0％鸡胆汁(或0.5％猪胆盐)的脱囊介质的作用而引起子孢子脱囊。在41℃条件下,脱囊约需0.5—1.5小时。脱囊前后的子孢子非常活跃,从斯氏体消失处逸出,在孢子囊空壳中留下一明显残体。     

柔嫩艾美耳球虫安徽肥西(AHFX)株的分离与鉴定

目的 分离并鉴定安徽肥西病鸡盲肠内的柔嫩艾美耳球虫.方法 通过雏鸡进行单卵囊接种与增殖试验,对所获卵囊用形态学方法进行初步鉴定;运用PCR方法扩增该分离株的ITS-1基因序列,并与相关序列进行比对、构建系统进化树.结果 该球虫分离株孢子化卵囊的平均大小为21.1 μm ×18.0 μm,卵形指数为1.17,潜隐期为140 h;其ITS-1基因序列与柔嫩艾美耳球虫(Eimeria tenella)GQ856310相似性达98.2％,二者的亲缘关系非常接近.结论 该球虫分离株为柔嫩艾美耳球虫(E.tenella),暂命名为柔嫩艾美耳球虫安徽肥西(AHFX)株.     

柔嫩艾美耳球虫早熟系的选育及其生物学特性研究

选育制备弱毒疫苗所需的柔嫩艾美耳球虫早熟系并对其生物学特性进行研究。运用Jeffers建立的早熟系选育方法对柔嫩艾美耳球虫山西株（E．tenella SX010323）进行早熟选育,并对其内生发育、致病性、繁殖力、免疫原性、稳定性进行观察和检测。结果表明：经过海兰白雏鸡15次传代之后,E．tenella SX010323潜隐期由141h缩短至120h,卵囊明显缩小。选育得到的柔嫩艾美耳球虫山西株早熟系（E．tenella SX010323P15）其内生发育表现第二代裂殖生殖不完全;亲本株与早熟系对11日龄雏鸡的半数致死量分别为7．52×10^4个／只、27．64×10^4个／只,早熟系在致病性与繁殖力上均较母株大幅度降低,但保留了母株的免疫原性,经早熟系免疫的雏鸡可抵抗亲本株半数致死量的攻击。对选育的早熟系进行放松选择传代10次,其潜隐期、OPG、致病性均保持了早熟系的特征。提示通过早熟选育得到了遗传相对稳定的柔嫩艾美耳球虫早熟系。     

Synaptonemal complex karyotype of Eimeria tenella

In most organisms, biological variability rests on the behaviour of the chromosomes in the meiotic context. Despite the importance of meiosis, very little is known about the meiotic behaviour of the Eimeria chromosomes. The aim of the present study is to describe the standard synaptonemal complex karyotype from Eimeria tenella oocyst spreads by electron microscopy. For that purpose, complete sets of pachytene synaptonemal complexes were obtained and the morphological pachytene karyotype was determined. The authors used a previously reported method that overcomes the difficulty of the extreme resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The chromosomes were selected under a light microscope and those selected were stained with phosphotungtic acid and studied by transmission electron microscopy. The authors confirmed 14 chromosomes, which were observed as synaptonemal complexes, and the karyotype was constructed by arranging synaptonemal complexes according to their relative lengths and kinetochore position. Components of the synaptonemal complex, lateral elements, central element, recombination nodules and kinetochore were observed. Measures of the kynetochore, width of the synaptonemal complex, diameter of the recombination nodule and length of the telomeres are given. Minimal and no significant differences were found between measures of chromosomes isolated from different Eimeria tenella strains. To the best of our knowledge, the present investigation for the first time identifies and describes the morphological characteristics of the synaptonemal complex of Eimeria tenella during the meiosis that occurs within the oocysts. In addition, the authors provide evidence of the presence of recombination nodules, suggesting that the recombination process may play an important role in the molecular evolution of this parasite.     

柔嫩艾美耳球虫孢子化卵囊cDNA文库的构建

用建立表达性文库的方法 ,构建了Eimeriatenella孢子化卵囊噬菌体ZAP表达性cDNA文库。首先用TRIzol试剂盒从E .tenella孢子化卵囊中提取总RNA ,再用Oligo(dT)₁₂纤维素柱从总RNA中分离mRNA ,以mRNA为模板 ,Oligo(dT)₁₈Linker Primer为引物 ,反转录合成cDNA第一链 ,再在DNA聚合酶Ⅰ作用下置换合成第二链cDNA。cDNA第二链合成后用PfuDNA聚合酶补平XhoⅠ位点 ,再与EcoRⅠAdapters连接 ,经XhoⅠ酶切后 ,凝胶电泳回收 500bp～4.0kp之间的cDNA片段 ,纯化后的双链cDNA与载体ZAPExpressvector连接。体外包装后得到E .tenella孢子化卵囊的cDNA表达性文库 ,经测定该文库的容量为 6×10⁶ ,扩增后文库的滴度为 1×10¹¹ Pfu mL ,经PCR测定 ,该文库的重组率为 96%。     

Solution structure of a PAN module from the apicomplexan parasite Eimeria tenella

Micronemes, specialised organelles found in all apicomplexan parasites, secrete molecules that are essential for parasite attachment and invasion of host cells. EtMIC5 is one such microneme protein that contains eleven tandemly repeating modules. These modules have homology with the PAN module superfamily. Members of this family are found in blood clotting proteins, some growth factors and some nematode proteins. This paper presents the structure of the 9^th PAN module in EtMIC5, determined using high resolution NMR. The structure shows similarities to and some differences from the N-terminal module of hepatocyte growth factor (HGF), the only previous member of the PAN family with known structure. AbbreviationsNMR – nuclear magnetic resonance; NOE – nuclear Overhauser enhancement; NOESY – NOE spectroscopy; COSY – correlated spectroscopy; TOCSY – total correlated spectroscopy; HSQC – hetero nuclear single quantum coherence; HMQC-J – hetero nuclear multiple quantum coherence-J coupling; MICs – microneme proteins; EtMIC5 – a microneme protein from Eimeria tenella; Apple9 – the ninth Apple repeat of EtMIC5; FXI – blood coagulation factor XI; PK – plasma prekallikrein; HGF – hepatocyte growth factor.     

柔嫩艾美耳球虫(Eimeria tenella)早熟株与亲本株致病性和繁殖力的研究

通过对经15代选育的柔嫩艾美耳球虫(E. tenella)山西株的早熟株与其亲本株的繁殖力和致病性进行比较研究,证实早熟株的潜隐期比亲本株缩短21 h,繁殖力下降40%左右;对致病性的研究显示,早熟株感染后对鸡只增重、AC I的影响较小,对11日龄雏鸡的半数感染量和半数致死量较亲本株增大,肠道病变记分较亲本株下降。由此认为,该早熟株符合球虫早熟株的特性,可用于鸡球虫病早熟苗的制作。     

Dihydrofolate Reductase from Eimeria tenella: Rationalization of Chemotherapeutic Efficacy of Pyrimethamine

SYNOPSIS. Dihydrofolate reductase activity of 0.2 nmole of dihydrofolate reduced/min/mg protein was detected in crude extracts of unsporulated oocysts of Eimeria tenella. The enzyme was purified by a combination of affinity and ion-exchange chromatography. Its molecular weight was estimated as 240,000 daltons. An anticoccidial drug pyrimethamine is a potent inhibitor of the activity of E. tenella dihydrofolate reductase ( K _t = 3 nM), but it is less effective an inhibitor of dihydrofolate reductase from chicken liver. This difference may explain the in vivo therapeutic action of pyrimethamine against Coccidia.     

Scanning Electron Microscopy of Schizogony in Eimeria tenella

SYNOPSIS. Developing 2nd- and 3rd-generation schizonts of Eimeria tenella were found in the ceca of chicks infected orally with sporulated oocysts. Several free 2nd-generation schizonts, which varied in diameter from 11 to 21.6 μm, were found on the epithelial surface of the cecum. Some schizonts appeared to have lost merozoites. Other schizonts were intact, one of which was surrounded by an unbroken membrane that followed the contours of the merozoites. Third-generation schizonts, much smaller than 2nd-generation schizonts and with fewer merozoites, were found only on cut or fractured surfaces of the cecal tissue. Third-generation merozoites appeared shorter and thicker than those of the 2nd-generation and were attached to the schizont residuum. A form with conical protuberances and another with 4 triangular segments were found; they were believed to be developing stages 3rd-generation schizonts.     

Mannitol metabolism in Eimeria tenella.

, and 1992. Mannitol metabolism in Eimeria tenella. International Journal for Parasitology 22: 1157–1163. Unsporulated oocysts of Eimeria tenella contain large quantities of carbohydrates, namely amylopectin, mannitol and glucose. Analysis of the carbohydrate content of sporulating oocysts revealed that mannitol content increased markedly during early stages of sporogony (first 4–6 h) but slowly diminished during the next 40 h of sporulation. Accumulation of mannitol was accompanied by a rapid decrease in amylopectin and free glucose, suggesting that mannitol might be synthesized from glucose released from amylopectin. Mannitol was also detected in sporozoite and merozoite extracts. All four mannitol cycle enzymes were detected in oocysts. Sporozoites excysted in vitro had lower activities of all four enzymes. Mannitol-1 -phosphatase and mannitol dehydrogenase activity was also detected in merozoites obtained from the second stage schizonts. Sporozoites incubated with ¹⁴C-glucose accumulated radioactively labelled precursor continuously for over 12 h and some of the ¹⁴C-glucose was converted into ¹⁴C-mannitol. These results indicate that mannitol plays an important role in the metabolism and development of the intracellular stages of the parasite.     

Comparative Development of Eimeria tenella from Sporozoites to Oocysts in Primary Kidney Cell Cultures from Gallinaceous Birds

Eimeria tenella completed its endogenous life cycle in primary cultures of kidney cells from 2- to 3-week-old-chickens, guinea fowl, partridges, pheasants, quail, and turkeys. Similarity in percentage of infection at 4 hr suggested that sporozoites entered cells from all birds in equal numbers. Development was better, however, in chicken cells in that the percentage of survival and of developmental stages during the first 2 days were greater, developmental stages occurring after 2 days usually were found earlier, mature 2nd-generation schizonts and oocysts were larger, and oocyst production was far greater than in nonhost cells. Multinucleate macrogametes, which sometimes reached sizes 3–4 times greater than normal oocysts, are reported for the first time.     

A survey of genes in Eimeria tenella merozoites by EST sequencing

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.