朊蛋白核酸疫苗免疫效果初步评价

发布时间：2018-02-20 23:17

本文关键词： 朊病毒病 DNA疫苗重组蛋白抗体细胞免疫　出处：《安徽理工大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的将带有泛素、溶酶体及内质网信号的PRNP真核表达载体作为DNA疫苗免疫小鼠,以期打破机体对朊病毒的免疫耐受,获得较好的体液免疫和细胞免疫反应,为朊病毒病的疫苗性免疫预防提供一定的数据和参考。方法(1)PRNP及带细胞表达定位信号的PRNPDNA疫苗,瞬时转染HeLa细胞后,使用Western Blot检测蛋白表达特点。(2)采取DNA肌肉注射与重组蛋白腹部皮下注射相结合的疫苗免疫方案,免疫结束后两周,采集小鼠血清、脾细胞,使用ELISA和ELISPOT方法检测血清IgG抗体和小鼠分泌y-IFN的脾细胞。将4-6周的雌性BALB/c小鼠随机分为6组,7只/组：pcDNA3.1空质粒组,pcDNA3.1-PrP组,pcDNA3.1-UbPrP组,pcDNA3.1-PrPLII组,pcDNA3.1-PrPER组,以上各实验组均进行蛋白增强免疫；另一组为pcDNA3.1不进行蛋白增强的对照组。无菌PBS稀释DNA疫苗至1μg/μl,小鼠左右腓肠肌注射各50μl/次,即100μg/次/只。DNA疫苗免疫三次后,实验组小鼠腹部皮下注射原核表达的重组PrP加强免疫两次,每次100μg。每两次免疫间隔两周,最后一次免疫后两周采集小鼠血清、脾细胞,进行免疫学检测。结果(1)各组质粒转染HeLa细胞后,分别于36小时和48小时检测载体的表达,实验结果显示各组疫苗质粒均能在HeLa细胞中表达,从而在25-35kD处均出现明显的被PrP单抗识别的特异性蛋白条带,且均具有三种糖基化类型,但表达量及蛋白量随时间的变化趋势却不尽相同。pcDNA3.1-PrP表达量最多,且48h时比36h蛋白量增多；pcDNA3.1-UbPrP在36h时表达的PrP以双糖基化为主随时间延长,蛋白降解明显；而pcDNA3.1-PrPLII载体以单糖基化PrP蛋白表达为主,随着时间延长,无糖基化蛋白观察不到,pcDNA3.1-PrPLII载体表达的蛋白与其他组载体相比降解速度快,具有表达时间依赖性。pcDNA3.1-PrPER编码的蛋白双糖基化PrP量较少,而无糖基化形式所占比例明显增加。(2)血清IgG抗体测定DNA疫苗pcDNA3.1-PrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPLII、pcDNA3.1-PrPER、pcDNA3.1免疫3次后,蛋白增强2次,pcDNA3.1对照组不进行蛋白增强。结果显示核酸疫苗pcDNA3.1-PrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPLII和pcDNA3.1-PrPER组均可以诱导BALB/c小鼠产生特异性抗体,其滴度依次分别达到1：6400、1：6400、1：6400、1：3200。pcDNA3.1组经蛋白增强免疫后,抗体滴度也可达到1：200。以上各组血清均能有效地特异性识别重组全长PrP。(3) ELISPOT方法检测细胞免疫反应免疫结束,取小鼠脾细胞,使用PrP23-90、PrP91-231和PrP23-231蛋白刺激,结果发现PrP23-90蛋白刺激pcDNA3.1-PrPER组、pcDNA3.1-PrP组和pcDNA3.1-UbPrP组产生的y-IFN细胞数较多,依次为720/106、538/106和482/106。PrP23-231蛋白刺激pcDNA3.1-PrPER组、pcDNA3.1-PrPLII和pcDNA3.1-UbPrP组刺激产生γ-IFN细胞数依次为483/106、274/106和261/106。而PrP91-231蛋白刺激产生γ-IFN细胞数均小于50/106,提示朊蛋白刺激机体产生细胞免疫的免疫原表位定位于朊蛋白N端23-90aa区间。结论各种朊病毒DNA疫苗可有效打破机体免疫耐受,使免疫小鼠产生了有效的特异性体液免疫反应和细胞免疫反应。DNA疫苗与重组蛋白联合免疫,可一定程度提高免疫水平。图[10]表[10]参[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]

【学位授予单位】：安徽理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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