本文關(guān)鍵詞： 朊病毒病 DNA疫苗 重組蛋白 抗體 細(xì)胞免疫　出處：《安徽理工大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的將帶有泛素、溶酶體及內(nèi)質(zhì)網(wǎng)信號(hào)的PRNP真核表達(dá)載體作為DNA疫苗免疫小鼠,以期打破機(jī)體對(duì)朊病毒的免疫耐受,獲得較好的體液免疫和細(xì)胞免疫反應(yīng),為朊病毒病的疫苗性免疫預(yù)防提供一定的數(shù)據(jù)和參考。 方法(1)PRNP及帶細(xì)胞表達(dá)定位信號(hào)的PRNPDNA疫苗,瞬時(shí)轉(zhuǎn)染HeLa細(xì)胞后,使用Western Blot檢測(cè)蛋白表達(dá)特點(diǎn)。(2)采取DNA肌肉注射與重組蛋白腹部皮下注射相結(jié)合的疫苗免疫方案,免疫結(jié)束后兩周,采集小鼠血清、脾細(xì)胞,使用ELISA和ELISPOT方法檢測(cè)血清IgG抗體和小鼠分泌y-IFN的脾細(xì)胞。將4-6周的雌性BALB/c小鼠隨機(jī)分為6組,7只/組：pcDNA3.1空質(zhì)粒組,pcDNA3.1-PrP組,pcDNA3.1-UbPrP組,pcDNA3.1-PrPLII組,pcDNA3.1-PrPER組,以上各實(shí)驗(yàn)組均進(jìn)行蛋白增強(qiáng)免疫；另一組為pcDNA3.1不進(jìn)行蛋白增強(qiáng)的對(duì)照組。無(wú)菌PBS稀釋DNA疫苗至1μg/μl,小鼠左右腓腸肌注射各50μl/次,即100μg/次/只。DNA疫苗免疫三次后,實(shí)驗(yàn)組小鼠腹部皮下注射原核表達(dá)的重組PrP加強(qiáng)免疫兩次,每次100μg。每?jī)纱蚊庖唛g隔兩周,最后一次免疫后兩周采集小鼠血清、脾細(xì)胞,進(jìn)行免疫學(xué)檢測(cè)。 結(jié)果(1)各組質(zhì)粒轉(zhuǎn)染HeLa細(xì)胞后,分別于36小時(shí)和48小時(shí)檢測(cè)載體的表達(dá),實(shí)驗(yàn)結(jié)果顯示各組疫苗質(zhì)粒均能在HeLa細(xì)胞中表達(dá),從而在25-35kD處均出現(xiàn)明顯的被PrP單抗識(shí)別的特異性蛋白條帶,且均具有三種糖基化類(lèi)型,但表達(dá)量及蛋白量隨時(shí)間的變化趨勢(shì)卻不盡相同。pcDNA3.1-PrP表達(dá)量最多,且48h時(shí)比36h蛋白量增多；pcDNA3.1-UbPrP在36h時(shí)表達(dá)的PrP以雙糖基化為主隨時(shí)間延長(zhǎng),蛋白降解明顯；而pcDNA3.1-PrPLII載體以單糖基化PrP蛋白表達(dá)為主,隨著時(shí)間延長(zhǎng),無(wú)糖基化蛋白觀察不到,pcDNA3.1-PrPLII載體表達(dá)的蛋白與其他組載體相比降解速度快,具有表達(dá)時(shí)間依賴(lài)性。pcDNA3.1-PrPER編碼的蛋白雙糖基化PrP量較少,而無(wú)糖基化形式所占比例明顯增加。(2)血清IgG抗體測(cè)定DNA疫苗pcDNA3.1-PrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPLII、pcDNA3.1-PrPER、pcDNA3.1免疫3次后,蛋白增強(qiáng)2次,pcDNA3.1對(duì)照組不進(jìn)行蛋白增強(qiáng)。結(jié)果顯示核酸疫苗pcDNA3.1-PrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPLII和pcDNA3.1-PrPER組均可以誘導(dǎo)BALB/c小鼠產(chǎn)生特異性抗體,其滴度依次分別達(dá)到1：6400、1：6400、1：6400、1：3200。pcDNA3.1組經(jīng)蛋白增強(qiáng)免疫后,抗體滴度也可達(dá)到1：200。以上各組血清均能有效地特異性識(shí)別重組全長(zhǎng)PrP。(3) ELISPOT方法檢測(cè)細(xì)胞免疫反應(yīng)免疫結(jié)束,取小鼠脾細(xì)胞,使用PrP23-90、PrP91-231和PrP23-231蛋白刺激,結(jié)果發(fā)現(xiàn)PrP23-90蛋白刺激pcDNA3.1-PrPER組、pcDNA3.1-PrP組和pcDNA3.1-UbPrP組產(chǎn)生的y-IFN細(xì)胞數(shù)較多,依次為720/106、538/106和482/106。PrP23-231蛋白刺激pcDNA3.1-PrPER組、pcDNA3.1-PrPLII和pcDNA3.1-UbPrP組刺激產(chǎn)生γ-IFN細(xì)胞數(shù)依次為483/106、274/106和261/106。而PrP91-231蛋白刺激產(chǎn)生γ-IFN細(xì)胞數(shù)均小于50/106,提示朊蛋白刺激機(jī)體產(chǎn)生細(xì)胞免疫的免疫原表位定位于朊蛋白N端23-90aa區(qū)間。 結(jié)論各種朊病毒DNA疫苗可有效打破機(jī)體免疫耐受,使免疫小鼠產(chǎn)生了有效的特異性體液免疫反應(yīng)和細(xì)胞免疫反應(yīng)。DNA疫苗與重組蛋白聯(lián)合免疫,可一定程度提高免疫水平。 圖[10]表[10]參[69]
[Abstract]:Objective with ubiquitin, PRNP eukaryotic lysosomes and endoplasmic reticulum signal expression vector as DNA vaccine in mice, in order to break the immune tolerance of prions, obtain good humoral and cellular immune responses, provide certain data and reference for prion disease vaccine immunity prevention.
Methods (1) and PRNP cell expression and localization of signal PRNPDNA vaccine, transient transfection of HeLa cells, using Western Blot to detect protein expression characteristics. (2) take the vaccine immunization program DNA intramuscular injection of recombinant protein and abdominal subcutaneous injection combined, two weeks after the final immunization, spleen cells of mice serum was collected, and the use of ELISA and the ELISPOT method for detection of serum IgG antibody in mice and y-IFN secretion of spleen cells. Female BALB/c mice were randomly divided into 6 groups for 4-6 weeks, 7 rats in each group: pcDNA3.1 empty plasmid group, pcDNA3.1-PrP group, pcDNA3.1-UbPrP group, pcDNA3.1-PrPLII group, pcDNA3.1-PrPER group, the experimental groups were enhanced protein immunity; another group pcDNA3.1 of control group. PBS protein enhanced sterile dilution DNA vaccine to 1 mu g/ Mu L, the left and right mouse gastrocnemius muscle injection of 50 l/ each time, namely 100 g/ /.DNA vaccine after three times, the experimental group of mice abdominal subcutaneous The recombinant PrP injected with prokaryotic expression was strengthened for two times, 100 g. each time, two times every two times, and two weeks after the last immunization, the serum and spleen cells were collected for immunological detection.
Results (1) each plasmid was transfected into HeLa cells, the expression of 36 hours and 48 hours respectively to detect the carrier, experimental results show that the group of vaccine plasmids were expressed in HeLa cells, resulting in 25-35kD were observed by PrP monoclonal antibody recognition of specific protein bands, and there are three kinds of glycosylation the type, but the expression quantity changes of protein content and the time is not the same expression of.PcDNA3.1-PrP and 48h than most, when the amount of 36h protein increased; the expression of pcDNA3.1-UbPrP in 36h PrP to disaccharide mainly with time prolonging, protein degradation is obvious; while the pcDNA3.1-PrPLII vector with the monosaccharide protein expression of PrP, with the time, non glycosylated protein observed, pcDNA3.1-PrPLII protein expression compared with other groups, carrier fast decomposition and protein PrP expression of disaccharide has time dependent.PcDNA3.1-PrPER encoding But no less glycosylated form proportion increased significantly. (2) serum IgG antibody DNA vaccine pcDNA3.1-PrP, pcDNA3.1-UbPrP, pcDNA3.1-PrPLII, pcDNA3.1-PrPER, pcDNA3.1 after 3 times of immunization enhanced protein 2, pcDNA3.1 protein in control group were not enhanced. Results showed that the nucleic acid vaccine pcDNA3.1-PrP, pcDNA3.1-UbPrP, pcDNA3.1-PrPLII and pcDNA3.1-PrPER group could induce BALB/c mice to produce specific antibody, the titers were up to 1:6400,1:6400,1:6400,1:3200.pcDNA3.1 by protein immunity, antibody titer can reach more than 200. 1: serum can effectively identify the specificity of recombinant full-length PrP. (3) ELISPOT method to detect cell immunologic reaction end, the mice spleen cells, using PrP23-90 and PrP91-231. PrP23-231 protein stimulation, the results showed that PrP23-90 protein stimulated pcDNA3.1-PrPER group, pcDNA3.1-PrP group and PC The number of y-IFN cells in group DNA3.1-UbPrP, followed by 720/106538/106 and 482/106.PrP23-231 protein stimulated pcDNA3.1-PrPER group, pcDNA3.1-UbPrP group and pcDNA3.1-PrPLII stimulates the production of gamma -IFN cell number were 483/106274/106 and 261/106. and PrP91-231 protein stimulates the production of gamma -IFN cell number was less than 50/106, suggesting that the immune prion protein induce cell immune epitope located in prion protein the N end of the 23-90aa range.
Conclusion all kinds of prion DNA vaccine can effectively break the body immune tolerance, and produce effective specific humoral immune response and cellular immune response in immune mice..DNA vaccine combined with recombinant protein can improve immune level to some extent.
Figure [10] table [10], [69]

【學(xué)位授予單位】：安徽理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 王慶敏,胡振林,孫樹(shù)漢;結(jié)核桿菌保護(hù)性抗原-遍在蛋白質(zhì)系統(tǒng)的建立[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2003年01期

2 吳慧娟;張志剛;;泛素-蛋白酶體途徑及意義[J];國(guó)際病理科學(xué)與臨床雜志;2006年01期

3 何洪智;趙曉航;張立勇;吳e，

本文編號(hào)：1520311