本文選題：朊蛋白　切入點(diǎn)：定位表達(dá)　出處：《安徽理工大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 目的(1)構(gòu)建定位于溶酶體、泛素的PrP表達(dá)載體,并進(jìn)行蛋白表達(dá)特點(diǎn)及定位的鑒定;(2)動(dòng)物免疫后,初步研究此融合表達(dá)載體在小鼠體內(nèi)激起的細(xì)胞免疫反應(yīng)和體液免疫反應(yīng),得到一組最基礎(chǔ)的免疫數(shù)據(jù),為以后朊蛋白疫苗的研究提供基礎(chǔ)數(shù)據(jù)。 方法(1)將泛素基因、溶酶體膜定位信號(hào)序列基因和PrP基因連接,克隆至pcDNA3.1載體中,構(gòu)建表達(dá)載體pcDNA3.1-UPrP、pcDNA3.1-PrPL;瞬時(shí)轉(zhuǎn)染真核表達(dá)細(xì)胞,經(jīng)Western Blot和間接免疫熒光技術(shù)檢測(cè)PrP表達(dá)特點(diǎn);(2)小鼠分八組,其中四組小鼠按0、14、28天分三次分別用質(zhì)粒pcDNA3.1、pcDNA3.1-HuPrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPL免疫;另四組小鼠中,有一組按0、14、28天分三次免疫重組PrP,另三組分別用質(zhì)粒pcDNA3.1-HuPrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ首次免疫,PrP蛋白加強(qiáng)兩次。最后一次免疫后2周,取脾臟及血液進(jìn)行ELISPOT、ELISA、WesternBlot檢測(cè)。 結(jié)果(1)構(gòu)建的各種PrP定位表達(dá)載體均可定位表達(dá)具有三種類(lèi)型的糖基化分子的PrP,以雙糖基化分子類(lèi)型最多。帶有泛素、溶酶體信號(hào)尾的質(zhì)粒pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ的PrP表達(dá)隨著時(shí)間的延長(zhǎng)蛋白表達(dá)量下降,提示泛素、溶酶體信號(hào)能加速表達(dá)的PrP在細(xì)胞內(nèi)的降解。(2)動(dòng)物免疫后,重組蛋白增強(qiáng)免疫的四組小鼠脾淋巴細(xì)胞在全長(zhǎng)人PrP的刺激下,UbPrP+rPrP組有兩只小鼠的脾淋巴細(xì)胞分泌特異性IFN-γ,分別為215、50SFCs/106/PBMCs;PrP+rPrP組中有一只小鼠為113.5 SFCs/106/PBMCs,PrPLⅡ+rPrP組和rPrP組的小鼠脾淋巴細(xì)胞不分泌特異性IFN-γ;在DNA核酸疫苗免疫的四組小鼠脾淋巴細(xì)胞中,僅UbPrP組的淋巴細(xì)胞兩只小鼠淋巴細(xì)胞能產(chǎn)生明顯的斑點(diǎn),分別為56、50 SFCs/106/PBMC,其他組的脾淋巴細(xì)胞在重組蛋白的刺激下均為陰性。小鼠抗血清ELISA結(jié)果顯示,重組蛋白組免疫小鼠的抗血清的滴度為1∶51200,經(jīng)質(zhì)粒初免、重組蛋白增強(qiáng)小鼠抗血清效價(jià)在大于1∶102400,明顯高于單純重組蛋白免疫組;UbPrP組及PrPLⅡ組質(zhì)粒免疫小鼠抗血清滴度大于1∶4 00;未修飾PrP組及空載體pcDNA3.1組小鼠抗血清與免疫前血清的P/N值小于2.1。各組抗血清特異性檢測(cè)的WB結(jié)果顯示:各組免疫小鼠的抗血清均可特異性識(shí)別重組人PrP。 結(jié)論:成功構(gòu)建了溶酶體、泛素定位表達(dá)的PRNP核酸疫苗載體pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ;與泛素融合表達(dá)及定位于溶酶體的PrP能一定程度地打破免疫耐受,誘導(dǎo)特異性免疫反應(yīng)。
[Abstract]:Objective 1) to construct a PrP expression vector targeting lysosome and ubiquitin, and to identify the protein expression characteristics and localization. After immunizing animals, we preliminarily studied the cellular and humoral immune responses induced by the fusion expression vector in mice. A set of basic immune data was obtained to provide basic data for the further study of prion vaccine. Methods 1) the ubiquitin gene, lysosomal membrane localization signal sequence gene and PrP gene were ligated into the pcDNA3.1 vector to construct the expression vector pcDNA3.1-UPrPnpcDNA3.1-PrPL. Western Blot and indirect immunofluorescence technique were used to detect the expression of PrP in mice. The mice were divided into eight groups, four of them were immunized with pcDNA3.1-UbPrPsil-pcDNA3.1-UbPrPsil-pcDNA3.1-PrPL three times according to the day of 14 ~ (28) day, and the other four groups of mice were immunized with pcDNA3.1-UbPrPfP pcDNA3.1-PrPL, respectively. One group was immunized with recombinant PrP three times according to day 1428, and the other three groups were immunized twice with pcDNA3.1-UbPrPmP pDNA3.1-UbPrPL 鈪，

本文編號(hào)：1568998