新孢子虫GRA7基因与GRA2基因串联载体的构建与表达

发布时间：2018-07-06 10:29

本文选题：新孢子虫 + SOE-PCR　；参考：《延边大学》2017年硕士论文

【摘要】：新孢子虫病(Neosporiasis)是一种由犬新孢子虫(Neospora caninum)引起的全球性寄生虫病。属于顶复门,孢子虫纲,球虫亚纲,真球虫目,肉孢子虫科,新孢子虫属。主要寄生在宿主的中枢神经系统、肌肉、肝、脑及其它内脏组织。该病于1984年首次在挪威发现,于1988年被Dubey博士命名为犬新孢子虫。新孢子虫的终末宿主:犬和狐狸;中间宿主:牛、羊、马、猪、兔等,该病主要引起孕畜的流产或死胎,以及新生儿的运动神经障碍,对牛的危害尤为严重,是引起牛流产的主要原因之一。该病呈世界性分布,广泛分布于欧洲等30余国家,部分牛群血清抗体的阳性率达到80%,我国奶牛的血清抗体阳性率为30%左右。因此有效控制新孢子虫病的发生是当务之急。本试验将新孢子虫的致密颗粒蛋白GRA7基因与GRA2基因通过柔性氨基酸连接,利用SOE PCR技术串联在一起,连接到表达载体中,表达出有良好免疫反应原性的重组蛋白。本试验从培养的新孢子虫Nc-1株提取RNA,反转录成cDNA,并以新孢子虫cDNA为模板,用NcGRA7的引物,进行第一次PCR扩增,得到大小为696 bp的目的片段;用NcGRA2的引物,进行第二次PCR扩增,得到大小为567bp的目的片段;应用SOE-PCR将两次PCR的产物拼接起来,得到串联基因NcGRA7-NcGRA2,大小为1263 bp。然后将该串联基因与pMD-19-T simple载体连接,经PCR鉴定,双酶切鉴定,测序鉴定,确定两个基因成功串联,将NcGRA7-NcGRA2基因片段和原核表达载体pGEX-4T-1连接,经Western blot分析,结果表明,重组原核表达载体成功表达串联蛋白。将NcGRA7-NcGRA2基因片段和真核表达载体pcDNA3.1连接,经IFAT鉴定,结果表明重组真核载体可以在哺乳动物细胞内表达外源蛋白。
[Abstract]:Neosporiasis is a global parasitic disease caused by Neospora caninum. Belongs to the phylum, sporidium, subclass coccidia, eucoccidae, meatosporidium, neosporidium. Parasitic mainly in the host central nervous system, muscle, liver, brain and other visceral tissues. The disease was first discovered in Norway in 1984 and named by Dr. Dubey as Neosporidium canis in 1988. The final hosts of neosporidium: dogs and foxes; intermediate hosts: cattle, sheep, horses, pigs, rabbits, etc. Is one of the main causes of cattle abortion. The disease is distributed all over the world. It is widely distributed in more than 30 countries such as Europe. The positive rate of serum antibody in some cattle reaches 80%. The positive rate of serum antibody in dairy cattle in China is about 30%. Therefore, it is urgent to control the occurrence of neosporidiosis effectively. In this experiment, GRA7 gene of neosporidium and GRA2 gene were linked with GRA2 gene by flexible amino acid, and then connected with each other by SOE PCR technique to express recombinant protein with good immunoreactivity. In this experiment, RNAs were extracted from cultured Nc-1 strain of Neosporidium, and transcribed into cDNAs. Using NcGRA7 primer as template, the target fragment of 696 BP was obtained by using NcGRA7 primer, and the second PCR amplification was carried out with the primers of NcGRA2, NcGRA2, NcGRA2, NcGRA2, NcGRA2, NcGRA2 and NcGRA7, respectively. The target fragment of 567bp was obtained by using SOE-PCR, and the tandem gene NcGRA7-NcGRA2 was obtained by using SOE-PCR. The size of NcGRA7-NcGRA2 was 1263 BP. Then the tandem gene was ligated with pMD-19-T simple vector. The two genes were identified by PCR, double enzyme digestion and sequencing. The NcGRA7-NcGRA2 gene fragment was linked with the prokaryotic expression vector pGEX-4T-1. The results of Western blot analysis showed that NcGRA7-NcGRA2 gene was linked with pGEX-4T-1. The recombinant prokaryotic expression vector successfully expressed tandem protein. NcGRA7-NcGRA2 gene fragment was ligated with eukaryotic expression vector pcDNA3.1 and identified by Ifat. The results showed that the recombinant eukaryotic vector could express foreign proteins in mammalian cells.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.723

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