猬迭宫绦虫27kDa半胱氨酸蛋白酶编码cDNA的克

发布时间：2017-12-31 18:13

本文关键词：猬迭宫绦虫27kDa半胱氨酸蛋白酶编码cDNA的克隆、表达及时空表达模式研究　出处：《贵州医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的分析猬迭宫绦虫27kDa半胱氨酸蛋白酶(Cysteine protease,CP)分子生物学特性,了解其在猬迭宫绦虫成虫和裂头蚴不同部位及裂头蚴不同感染时期的表达模式。方法1.猬迭宫绦虫27kDa CP的编码cDNA序列分析及克隆、表达。(1)运用瑞士生物信息学研究所的蛋白分析专家系统(Ex PASy)和美国国家生物技术信息中心(NCBI)等有关的生物信息学分析工具,预测和分析猬迭宫绦虫CP的结构与生物学功能。(2)构建猬迭宫绦虫CP基因原核表达重组质粒pET-28a(+)-SEP-CP,并在大肠杆菌Transetta(DE3)中诱导表达,对所获重组蛋白进行SDS-PAGE分析。2.裂头蚴CP基因时空表达模式的检查。(1)收集安顺地区野生王锦蛇体内猬迭宫绦虫裂头蚴。将64只昆明小鼠随机分成8组,每组8只,以5条裂头蚴/只感染小鼠,于感染后30 min~14 d各剖杀1组小鼠,收集感染小鼠体内的裂头蚴。(2)以10条/只感染3只幼犬,于感染后1个月剖杀幼犬,收集成虫。(3)以18S rRNA(18S核糖体RNA)为内参基因,采用实时定量PCR检测猬迭宫绦虫成虫和裂头蚴不同部位及不同感染时期裂头蚴CP基因的表达情况。结果1.猬迭宫绦虫27kDa CP的编码cDNA去信号肽序列长954bp,编码一个含317个氨基酸的蛋白多肽,属于Peptidase_C39_like超家族。理论分子量35669.9Da,等电点5.92。2.SDS-PAGE电泳分析表明,该基因在原核表达系统中得到高效表达,产物分子量大小为35kDa,诱导产物主要以包涵体形式存在。3.小鼠经口感染裂头蚴30 min后腹腔内查见穿过胃肠壁的裂头蚴;感染后30min~6 h,裂头蚴主要见于胃肠腔、胃肠壁和腹腔内;感染24 h,有少量的裂头蚴移行至小鼠皮下肌肉组织;感染7 d后,多数裂头蚴移行至皮下肌肉组织寄生。4.实时荧光定量PCR检测猬迭宫绦虫27kDa CP基因在成虫及裂头蚴的不同部位均有表达,但成虫不同部位的表达量均低于幼虫时期。成虫不同部位的表达量依次为孕节头节成节,裂头蚴不同部位的表达量依次为体段头段尾段。在感染30min时,裂头蚴的27kDa CP基因表达量上调,其表达量是未感染时的2.1倍,随后表达量呈下调,至6h表达量最低,随后27kDa CP基因表达量呈上升趋势,至14d时达到峰值。结论1.猬裂头蚴27kDa CP属于Peptidase_C39_like超家族。2.成功构建了pET-28a(+)-SEP-CP原核表达体系。3.初步揭示了27kDa CP基因在猬迭宫绦虫成虫及裂头蚴的时空表达模式,提示27kDa CP基因可能参与了虫体的入侵、移行、营养吸收、生长发育及生殖等过程。
[Abstract]:Objective to analyze the molecular biological characteristics of 27kDa cysteine protease (Cysteine protease) from Taenia hedgehoi. Objective: to investigate the expression patterns of Taenia hedgehoi in different parts of adult and mitococcus and at different infection stages. 1. The coding cDNA sequence of 27kDa CP of Taenia hedgehoi was analyzed and cloned. Expression 1) using the relevant bioinformatics analysis tools, such as the protein analysis expert system of the Swiss Bioinformatics Institute, PaiEx, and the National Biotechnology Information Center (NCBII) of the United States. The structure and biological function of CP were predicted and analyzed.) the prokaryotic expression plasmid pET-28a (pET-28a- SEP-CP) of Taenia hedgehog CP gene was constructed. The expression was induced in E. coli Transettade3. The recombinant protein was analyzed by SDS-PAGE. 2. Detection of the temporal and spatial expression pattern of CP gene in mitomycaria. A total of 64 Kunming mice were randomly divided into 8 groups. Eight mice in each group were infected with 5 caterpillars per mouse. Each group was killed 30 min~14 after infection. The mice were collected and infected with 10 caterpillars / 3 puppies. 1 month after infection, the puppies were killed and adult worms were collected. The 18s rRNA(18S ribosomal RNAs were used as internal reference genes. Real time quantitative PCR was used to detect the expression of CP gene in different parts and different infection stages of Taenia hedgehog adults and mitosis cercariae. Results 1.The expression of CP gene of Hymenia hedgehoi was 27 kDa. The encoding cDNA of CP was 954 BP in length. It encodes a protein polypeptide containing 317 amino acids, belonging to the Peptidase_C39_like superfamily. The theoretical molecular weight is 35669.9Da. The electrophoretic analysis of isoelectric point 5.92.2.SDS-PAGE showed that the gene was highly expressed in prokaryotic expression system and the molecular weight of the product was 35kDa. The induction product mainly existed in the form of inclusion body. After 30 min of mouse oral infection of mitomycaria, the mitomycaria through the gastrointestinal wall was found in the abdominal cavity. At 30 min and 6 h after infection, mitomycaria was mainly found in gastrointestinal cavity, gastrointestinal wall and abdominal cavity. After 24 hours of infection, a small number of mitomycaria was transferred to the subcutaneous muscle tissue of mice. After 7 days of infection, the majority of mitomycaria migrated to the subcutaneous muscle tissue. 4. Real-time fluorescence quantitative PCR was used to detect the expression of 27kDa CP gene in the adult and the different parts of the cysticercus. However, the expression of different parts of adult worm was lower than that of larval stage. The expression of different parts of adult worm was nodal in turn, and the expression of different parts of mitomycaria was at the end of head segment of body segment in turn. After 30 minutes of infection, the expression level of different parts of adult worm was the same as that of the tail of head segment of body segment. The expression of 27kDa CP gene in mitomycaria was up regulated, which was 2.1 times higher than that in the uninfected group, then decreased, and reached the lowest level at 6 h. Then the expression of 27kDa CP gene increased. Conclusion 1. The 27kDa CP belongs to the Peptidase_C39_like superfamily. 2. The pET-28a () was successfully constructed. The prokaryotic expression system of -SEP-CP. 3.The expression pattern of 27kDa CP gene in the adult and mitomycaria of Taenia hedgehoi was preliminarily revealed. These results suggest that the 27kDa CP gene may be involved in the invasion, migration, nutrient absorption, growth, development and reproduction of the insect.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R383.3

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