旋毛虫期特异性基因转录及表达特性研究

发布时间：2018-05-05 09:39

本文选题：旋毛虫 + 期特异性基因　；参考：《吉林大学》2007年硕士论文

【摘要】： 由于旋毛虫存在三个不同的发育时期,而各个发育时期具有不同的抗原表达,因此研究旋毛虫期特异性基因有助于探明旋毛虫侵袭、致病机理以及包囊形成的分子机制。我们利用分子生物学和免疫学方法对本室业已发现的2条新生幼虫期特异性(即旋毛虫包囊形成时期)基因——II型脱氧核糖核酸酶基因T314和丝氨酸蛋白酶基因T668进行了转录及表达特性研究,并探讨了其参与旋毛虫寄生及包囊形成过程中的可能机制。根据T314和T668 cDNA序列设计特异引物,从感染旋毛虫的小鼠骨骼肌中提取总RNA,应用RT-PCR技术分别反转录出T314和T668 cDNA,在分子水平上对这2个基因的转录时空进行了鉴定。结果显示:T314、T668基因均从新生幼虫出生即开始转录,随着日龄的增加mRNA的量逐渐减少,T314 mRNA在感染后第20天仍能检测到,而T668 mRNA于感染后11-14天转录趋于停止。分别利用本室制备的兔抗T314和T668重组蛋白抗体,采用免疫组化技术,观察T314和T668基因编码蛋白的组织学定位。结果表明:T314、T668蛋白在新生幼虫感染后第1天均有表达,T314蛋白分布于虫体表皮,而T668蛋白分布于虫体的杆状体区域;T314蛋白在感染后7-20天分泌到虫体外的保姆细胞细胞质上,而T668蛋白则在感染后6-13天分泌到虫体外肿大的肌细胞核上。该结果预示着:T314蛋白(即DNase II)可能与旋毛虫寄生所需要厌氧环境的形成有关,可能的机制为DNase II降解线粒体DNA,使包囊内的有氧代谢转变为无氧代谢;而T668蛋白(即丝氨酸蛋白酶)可能参与包囊的形成,其可能的机制是作为转录因子调控肿大的肌细胞核大量表达胶原蛋白从而形成包囊。从pMD-18T-T314重组质粒中扩增T314目的片段,重组到PCR II载体中。利用SP6和T7 RNA聚合酶体外转录合成siRNA。通过浸泡将siRNA导入新生幼虫体内进行T314基因沉默,发现T314基因沉默后虫体总数减少,形成的包囊大部分空泡化,初步验证T314基因可能与旋毛虫包囊形成无关。
[Abstract]:Since there are three different developmental stages of Trichinella spiralis, and different antigens are expressed in each developmental stage, studying the specific genes of Trichinella spiralis is helpful to understand the invasion, pathogenesis and the molecular mechanism of cyst formation of Trichinella spiralis. We have used molecular biology and immunological methods to identify two new larval stage specific genes (I. e., the stage of cysts formation of Trichinella spiralis): type II deoxyribonuclease gene T314 and serine protease gene. The transcriptional and expression characteristics of T668 were studied. The possible mechanism of its involvement in parasitism and cyst formation of Trichinella spiralis was also discussed. According to the sequence of T314 and T668 cDNA, specific primers were designed to extract total RNAs from skeletal muscle of mice infected with Trichinella spiralis. T314 and T668 cDNAwere inverted by RT-PCR technique. The transcriptional time and space of these two genes were identified at molecular level. The results showed that the T314T668 gene was transcribed from the birth of the newborn larva. With the increase of age, the amount of T314 mRNA could be detected on the 20th day after infection, while the transcription of T668 mRNA tended to stop at 11-14 days after infection. Using rabbit anti-T314 and T668 recombinant protein antibodies prepared in our laboratory, the histological localization of T314 and T668 gene encoded proteins was observed by immunohistochemical technique. The results showed that T314T668 protein was expressed in the epidermis of the larva on the first day after infection, while the T668 protein was secreted into the cytoplasm of the nanny in vitro 7-20 days after infection. The T 668 protein was secreted 6-13 days after infection to the swollen muscle nucleus of the insect in vitro. The results suggest that the DNase II protein may be related to the formation of anaerobic environment required by Trichinella spiralis parasitism. The possible mechanism is that DNase II degrades mitochondrial DNA and changes aerobic metabolism into anaerobic metabolism in the cyst. T668 protein (serine protease) may be involved in the formation of cysts, and its possible mechanism is to regulate the expression of collagen protein in the enlarged muscle nucleus as a transcription factor. The target fragment of T314 was amplified from pMD-18T-T314 recombinant plasmid and recombined into PCR II vector. SiRNA was synthesized by in vitro transcription of SP6 and T7 RNA polymerase. SiRNA was introduced into the newborn larva for T314 gene silencing. It was found that the total number of body decreased and most of the cysts were vacuolated after the silencing of T314 gene. It was preliminarily verified that T314 gene may not be related to the formation of trichinella cyst.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383;S852.7

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