Molecular cloning, expression and immunohistochemical localization of Ac-ALMP, an astacin-like metalloprotease from Angiostrongylus cantonensis

广州管圆线虫病是一种人畜共患的寄生虫病。它是因人进食含有广州管圆线虫感染期幼虫的食物或水源而引起的。随着人们饮食习惯的改变和水陆交通的日趋发达，该病的流行呈逐年上升的趋势，目前尚无理想的特效药对该病进行防治。虾红素金属蛋白酶是金属蛋白酶家族的一员，研究发现，其在寄生虫寄生生活中发挥一系列不同的生物学功能。但是目前国际上，关于广州管圆线虫虾红素金属蛋白酶的报道几乎没有。 本研究为探索广州管圆线虫虾红素金属蛋白酶在虫体中的功能，对其基因进行调取，我们通过虾红素金属蛋白酶EST序列设计引物进行RACE克隆，最终获得广州管圆线虫虾红素金属蛋白酶全长基因，并将其命名为Ac-ALMP（Astacin-l...Angiostrongyliasis cantonensis is one of zoonotic parasitic disease. Human or animals can infect because consuming raw or undercooked intermediated hosts, contaminated vegetables. In recent years, the incidence of angiostrongyliasis has become increasingly significant with the change of diet habits along with development of transport facilities in water and land. However, there are not effective m...学位：理学硕士院系专业：生命科学学院_生物化学与分子生物学学号：2162013115459

r DNA sequence analysis and morphological redescription of Empoasca onukii from the tea growing area of Fujian

重新描述了小贯小绿叶蝉的形态结构,尤其是头部色斑、翅脉、腹部内突、下生殖板及其刚毛着生位置,并增加了足部(刺毛列)特征描述。通过PCr技术克隆得到该物种的r dnA序列,包括部分18S(1 843bP)和28S序列(667 bP)以及完整的5.8S(155 bP)、ITS1(3 114 bP)和ITS2序列(1 008 bP)。序列分析表明,18S,5.8S,28S与其他物种间具有90%~95%的序列一致性;而ITS1和ITS2序列变异性非常大。碱基组成比率分析显示ITS1以及ITS2具有AT偏好性,前者A+T占66.0%,后者占65.1%。与亲缘种的比较分析显示ITS1和ITS2具有丰富的多态位点,并且ITS2更适用于近缘种的分子鉴定。The morphological structures of Empoasca onukii,the cephalic splashes,vein,abdominal apodemes,subgenital plate and the arrangement of its bristles,and the characteristics of legs were redescribed,especially the arrangement of spines were added.Ribosomal DNA( r DNA) including partial of 18S( 1 843 bp),28S( 667 bp) and complete 5.8S( 155 bp),ITS1( 3 114 bp),ITS2( 1 008 bp) were isolated by PCR technology.Sequence analysis indicated that 18 S,5.8S and 28 S had high sequence identity( 90%- 95%) with other insects; however,ITS1 and ITS2 showed much variability.The analysis of base composition showed that ITS1 and ITS2 were more preferences for AT( contained 66.0% and 65.1% A + T,respectively).Comparison with the phylogenetic species revealed that ITS1 and ITS2 from E.onukii had rich polymorphic loci and ITS2 was more suitable for molecular identification of closely related species.国家自然科学基金(81171595

The multilayered Zhelvak 5 site in Northern Tuva and its surroundings : geomagnetic surveys and initial research findings

W artykule przedstawiono pierwsze wyniki badań terenowych na stanowisku osadowym Żelwak 5 w Tuwie, w dolinie Turano-Ujukskiej, datowanym na okres epoki żelaza, oraz na podobnych obiektach znajdujących się w jego otoczeniu. Wyodrębnione kompleksy ceramiczne wskazują na długotrwały okres funkcjonowania tego stanowiska. Wyniki badań geomagnetycznych na trzech obiektach ujawniły potencjalne obiekty archeologiczneThe article covers the initial results of field research conducted at the Zhelvak 5 settlement in Tuva, Turan-Uyuk Valley, dated to Early Iron Age, and similar locations within its vicinity. The identified ceramic assemblages suggest a prolonged period of activity at the site. Additionally, geomagnetic surveys at three sites revealed potential archaeological featuresВ статье освещены первые результаты полевых исследований на стоянке раннего железного века Желвак 5 в Туве и на аналогичных объектах, расположенных в ее окружении. Выделенные керамические комплексы указывают на длительный период функционирования стоянки. А результате геомагнитных разведок на трех памятниках выявлены потенциальные археологические объект

Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine

Comparative transcriptomic analysis of two important life stages of Angiostrongylus cantonensis: fifth-stage larvae and female adults

Abstract The mechanisms involved in the fast growth of Angiostrongylus cantonensis from fifth-stage larvae (L5) to female adults and how L5 breaks through the blood-brain barrier in a permissive host remain unclear. In this work, we compared the transcriptomes of these two life stages to identify the main factors involved in the rapid growth and transition to adulthood. RNA samples from the two stages were sequenced and assembled de novo. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of 1,346 differentially expressed genes between L5 and female adults was then undertaken. Based on a combination of analytical results and developmental characteristics, we suggest that A. cantonensis synthesizes a large amount of cuticle in L5 to allow body dilatation in the rapid growth period. Products that are degraded via the lysosomal pathway may provide sufficient raw materials for cuticle production. In addition, metallopeptidases may play a key role in parasite penetration of the blood-brain barrier during migration from the brain. Overall, these results indicate that the profiles of each transcriptome are tailored to the need for survival in each developmental stage

Initial events in the breakthrough of the epithelial barrier of the small intestine by Angiostrongylus cantonensis

Although the third-stage larvae of Angiostrongylus cantonensis (AcL3) are thought to initiate infection by penetrating the epithelium of the small intestine, the mode of intestinal invasion remains obscure. Considering the inaccessibility of the gut tract and the need to sacrifice animals for this type of study, we devised an in vitro cell-parasite co-culture system to examine the initial cellular and molecular events between AcL3 and host epithelia. No apoptosis augmentation was detected in enterocytes after introduction of larvae. A significant increase in dead cells was detected in IEC-6, NCM460 and 293T after incubating for 4 h, with AcL3 wounding rat small intestinal epithelial cells IEC-6 more rapidly. Under a scanning electron microscope (SEM), cell gap opening was visualized in the IEC-6 monolayer treated with AcL3. Loosening of the extracellular matrix (ECM) of the monolayer was found to be involved in the parasite-cell interactions. Pretreating the AcL3 with a protease inhibitor attenuated its penetration ability of the artificial intestine barrier. In conclusion, AcL3 broke through the intestinal barrier of the host with the assistance of mechanical injury and the opening of a cell gap, but without causing apoptosis. The interaction platform presented here may provide direct insight into the cellular and molecular events during worm invasion of host enterocytes