本文關(guān)鍵詞：猬迭宮絳蟲(chóng)27kDa半胱氨酸蛋白酶編碼cDNA的克隆、表達(dá)及時(shí)空表達(dá)模式研究　出處：《貴州醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 猬迭宮絳蟲(chóng) 裂頭蚴 半胱氨酸蛋白酶 克隆表達(dá) 序列分析 時(shí)空表達(dá)

【摘要】：目的分析猬迭宮絳蟲(chóng)27kDa半胱氨酸蛋白酶(Cysteine protease,CP)分子生物學(xué)特性,了解其在猬迭宮絳蟲(chóng)成蟲(chóng)和裂頭蚴不同部位及裂頭蚴不同感染時(shí)期的表達(dá)模式。方法1.猬迭宮絳蟲(chóng)27kDa CP的編碼cDNA序列分析及克隆、表達(dá)。(1)運(yùn)用瑞士生物信息學(xué)研究所的蛋白分析專家系統(tǒng)(Ex PASy)和美國(guó)國(guó)家生物技術(shù)信息中心(NCBI)等有關(guān)的生物信息學(xué)分析工具,預(yù)測(cè)和分析猬迭宮絳蟲(chóng)CP的結(jié)構(gòu)與生物學(xué)功能。(2)構(gòu)建猬迭宮絳蟲(chóng)CP基因原核表達(dá)重組質(zhì)粒pET-28a(+)-SEP-CP,并在大腸桿菌Transetta(DE3)中誘導(dǎo)表達(dá),對(duì)所獲重組蛋白進(jìn)行SDS-PAGE分析。2.裂頭蚴CP基因時(shí)空表達(dá)模式的檢查。(1)收集安順地區(qū)野生王錦蛇體內(nèi)猬迭宮絳蟲(chóng)裂頭蚴。將64只昆明小鼠隨機(jī)分成8組,每組8只,以5條裂頭蚴/只感染小鼠,于感染后30 min~14 d各剖殺1組小鼠,收集感染小鼠體內(nèi)的裂頭蚴。(2)以10條/只感染3只幼犬,于感染后1個(gè)月剖殺幼犬,收集成蟲(chóng)。(3)以18S rRNA(18S核糖體RNA)為內(nèi)參基因,采用實(shí)時(shí)定量PCR檢測(cè)猬迭宮絳蟲(chóng)成蟲(chóng)和裂頭蚴不同部位及不同感染時(shí)期裂頭蚴CP基因的表達(dá)情況。結(jié)果1.猬迭宮絳蟲(chóng)27kDa CP的編碼cDNA去信號(hào)肽序列長(zhǎng)954bp,編碼一個(gè)含317個(gè)氨基酸的蛋白多肽,屬于Peptidase_C39_like超家族。理論分子量35669.9Da,等電點(diǎn)5.92。2.SDS-PAGE電泳分析表明,該基因在原核表達(dá)系統(tǒng)中得到高效表達(dá),產(chǎn)物分子量大小為35kDa,誘導(dǎo)產(chǎn)物主要以包涵體形式存在。3.小鼠經(jīng)口感染裂頭蚴30 min后腹腔內(nèi)查見(jiàn)穿過(guò)胃腸壁的裂頭蚴;感染后30min~6 h,裂頭蚴主要見(jiàn)于胃腸腔、胃腸壁和腹腔內(nèi);感染24 h,有少量的裂頭蚴移行至小鼠皮下肌肉組織;感染7 d后,多數(shù)裂頭蚴移行至皮下肌肉組織寄生。4.實(shí)時(shí)熒光定量PCR檢測(cè)猬迭宮絳蟲(chóng)27kDa CP基因在成蟲(chóng)及裂頭蚴的不同部位均有表達(dá),但成蟲(chóng)不同部位的表達(dá)量均低于幼蟲(chóng)時(shí)期。成蟲(chóng)不同部位的表達(dá)量依次為孕節(jié)頭節(jié)成節(jié),裂頭蚴不同部位的表達(dá)量依次為體段頭段尾段。在感染30min時(shí),裂頭蚴的27kDa CP基因表達(dá)量上調(diào),其表達(dá)量是未感染時(shí)的2.1倍,隨后表達(dá)量呈下調(diào),至6h表達(dá)量最低,隨后27kDa CP基因表達(dá)量呈上升趨勢(shì),至14d時(shí)達(dá)到峰值。結(jié)論1.猬裂頭蚴27kDa CP屬于Peptidase_C39_like超家族。2.成功構(gòu)建了pET-28a(+)-SEP-CP原核表達(dá)體系。3.初步揭示了27kDa CP基因在猬迭宮絳蟲(chóng)成蟲(chóng)及裂頭蚴的時(shí)空表達(dá)模式,提示27kDa CP基因可能參與了蟲(chóng)體的入侵、移行、營(yíng)養(yǎng)吸收、生長(zhǎng)發(fā)育及生殖等過(guò)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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R383.3

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