阴道毛滴虫长寿保障基因Tv-LAG1的克隆和表达及衰老相关基因筛选

发布时间：2018-07-09 19:07

  本文选题：阴道毛滴虫 + 衰老　； 参考：《汕头大学》2005年硕士论文

【摘要】：研究目的 (1)克隆原生动物阴道毛滴虫(Trichomonas vaginalis) 长寿保障基因(Longevity assurance gene 1 LAG1)同源基因:Tv- LAG1。构建Tv-LAG1原核表达重组体及表达其融合蛋白。(2)在 模式生物阴道毛滴虫cDNA消减杂交文库中,筛选和鉴定阴道毛 滴虫细胞周期及衰老相关基因。材料和方法 (1)从阴道毛滴 虫cDNA表达文库中筛选LAG1的同源基因,并对其序列保守结构 域进行同源性分析。用pET41表达载体与Tv-LAG1 cDNA克隆构建 原核表达重组体,在异丙基硫代-β-D-半乳糖苷(isopropylthio- β-D-galactoside IPTG)诱导下表达目的基因融合蛋白,Ni-NTA亲 和层析纯化表达产物,SDS-PAGE鉴定表达产物的分子量及纯化 效果。用纯化的融合蛋白免疫家兔,获得抗血清,采用Western blot 法对抗体特异性进行分析鉴定。(2)采用反转录PCR技术进一步 分析鉴定cDNA消减杂交文库中分离到的15个cDNA克隆。结果 (1)从阴道毛滴虫cDNA表达文库中成功筛选出长寿保障基因 LAG1的同源基因(Tv-LAG1)。其cDNA克隆含910对碱基,其编 码框长834 bp,推测蛋白质序列含有277个氨基酸。从植物到人 类的结构域序列分析提示,Tv-Lag1p与酵母Lag1p及其它同源蛋 白均有同源性。其中与拟芥兰相比,在258个氨基酸的序列区, 同源性达到23%。其次为人类Lag1p同源蛋白质,在216个氨基酸
[Abstract]:Objective (1) to clone the long assurance gene 1 LAG1 homologous gene of Trichomonas vaginalis. Construction of Tv-LAG1 prokaryotic expression recombinant and expression of its fusion protein. (2) screening and identification of trichomonas vaginalis cell cycle and senescence related genes in the model Trichomonas vaginalis cDNA subtractive hybridization library. Materials and methods (1) the homologous gene of LAG1 was screened from the cDNA expression library of Trichinella vaginalis and its conserved domain was analyzed. The prokaryotic expression recombinant was constructed by using pET41 expression vector and Tv-LAG1 cDNA clone. Isopropylthio- 尾 -D-galactoside (IPTG) was induced by isopropylthio- 尾 -Dgalactoside IPTG to express the target gene fusion protein: Ni-NTA affinity chromatography and SDS-PAGE to identify the molecular weight and purification effect of the expressed product. The antiserum was obtained by immunizing rabbits with the purified fusion protein, and the specificity of the antibody was analyzed by Western blot. (2) 15 cDNA clones isolated from the cDNA subtractive hybridization library were further analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Results (1) the homologous gene (Tv-LAG1) of longevity protection gene LAG1 was successfully screened from the cDNA expression library of Trichomonas vaginalis. The cDNA clone contained 910 pairs of bases, and its coding frame was 834 BP, which suggested that the protein sequence contained 277 amino acids. The analysis of the domain sequence from plant to human indicates that Tv-Lag1p shares homology with yeast Lag1p and other homologous egg whites. The homology of 258 amino acids was 23 in comparison with that of Brassica thaliana. The second is human Lag1p homologous protein, with 216 amino acids.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R383

【参考文献】

相关期刊论文 前1条

1 章家新,傅玉才,郑晓虹,刘红;阴道毛滴虫cDNA文库的构建和鉴定[J];汕头大学医学院学报;2003年04期

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本文编号：2110368