本文選題：新孢子蟲 + SOE-PCR��； 參考：《延邊大學(xué)》2017年碩士論文

【摘要】：新孢子蟲病(Neosporiasis)是一種由犬新孢子蟲(Neospora caninum)引起的全球性寄生蟲病。屬于頂復(fù)門,孢子蟲綱,球蟲亞綱,真球蟲目,肉孢子蟲科,新孢子蟲屬。主要寄生在宿主的中樞神經(jīng)系統(tǒng)、肌肉、肝、腦及其它內(nèi)臟組織。該病于1984年首次在挪威發(fā)現(xiàn),于1988年被Dubey博士命名為犬新孢子蟲。新孢子蟲的終末宿主:犬和狐貍;中間宿主:牛、羊、馬、豬、兔等,該病主要引起孕畜的流產(chǎn)或死胎,以及新生兒的運(yùn)動(dòng)神經(jīng)障礙,對(duì)牛的危害尤為嚴(yán)重,是引起牛流產(chǎn)的主要原因之一。該病呈世界性分布,廣泛分布于歐洲等30余國家,部分牛群血清抗體的陽性率達(dá)到80%,我國奶牛的血清抗體陽性率為30%左右。因此有效控制新孢子蟲病的發(fā)生是當(dāng)務(wù)之急。本試驗(yàn)將新孢子蟲的致密顆粒蛋白GRA7基因與GRA2基因通過柔性氨基酸連接,利用SOE PCR技術(shù)串聯(lián)在一起,連接到表達(dá)載體中,表達(dá)出有良好免疫反應(yīng)原性的重組蛋白。本試驗(yàn)從培養(yǎng)的新孢子蟲Nc-1株提取RNA,反轉(zhuǎn)錄成cDNA,并以新孢子蟲cDNA為模板,用NcGRA7的引物,進(jìn)行第一次PCR擴(kuò)增,得到大小為696 bp的目的片段;用NcGRA2的引物,進(jìn)行第二次PCR擴(kuò)增,得到大小為567bp的目的片段;應(yīng)用SOE-PCR將兩次PCR的產(chǎn)物拼接起來,得到串聯(lián)基因NcGRA7-NcGRA2,大小為1263 bp。然后將該串聯(lián)基因與pMD-19-T simple載體連接,經(jīng)PCR鑒定,雙酶切鑒定,測序鑒定,確定兩個(gè)基因成功串聯(lián),將NcGRA7-NcGRA2基因片段和原核表達(dá)載體pGEX-4T-1連接,經(jīng)Western blot分析,結(jié)果表明,重組原核表達(dá)載體成功表達(dá)串聯(lián)蛋白。將NcGRA7-NcGRA2基因片段和真核表達(dá)載體pcDNA3.1連接,經(jīng)IFAT鑒定,結(jié)果表明重組真核載體可以在哺乳動(dòng)物細(xì)胞內(nèi)表達(dá)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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.723

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