【作者】 李媛；

【导师】 王克霞；

【作者基本信息】 安徽理工大学 ， 病原生物学， 2009， 硕士

【摘要】 目的(1)构建定位于溶酶体、泛素的PrP表达载体,并进行蛋白表达特点及定位的鉴定;(2)动物免疫后,初步研究此融合表达载体在小鼠体内激起的细胞免疫反应和体液免疫反应,得到一组最基础的免疫数据,为以后朊蛋白疫苗的研究提供基础数据。方法(1)将泛素基因、溶酶体膜定位信号序列基因和PrP基因连接,克隆至pcDNA3.1载体中,构建表达载体pcDNA3.1-UPrP、pcDNA3.1-PrPL;瞬时转染真核表达细胞,经Western Blot和间接免疫荧光技术检测PrP表达特点;(2)小鼠分八组,其中四组小鼠按0、14、28天分三次分别用质粒pcDNA3.1、pcDNA3.1-HuPrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPL免疫;另四组小鼠中,有一组按0、14、28天分三次免疫重组PrP,另三组分别用质粒pcDNA3.1-HuPrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ首次免疫,PrP蛋白加强两次。最后一次免疫后2周,取脾脏及血液进行ELISPOT、ELISA、WesternBlot检测。结果(1)构建的各种PrP定位表达载体均可定位表达具有三种类型的糖基化分子的PrP,以双糖基化分子类型最多。带有泛素、溶酶体信号尾的质粒pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ的PrP表达随着时间的延长蛋白表达量下降,提示泛素、溶酶体信号能加速表达的PrP在细胞内的降解。(2)动物免疫后,重组蛋白增强免疫的四组小鼠脾淋巴细胞在全长人PrP的刺激下,UbPrP+rPrP组有两只小鼠的脾淋巴细胞分泌特异性IFN-γ,分别为215、50SFCs/106/PBMCs;PrP+rPrP组中有一只小鼠为113.5 SFCs/106/PBMCs,PrPLⅡ+rPrP组和rPrP组的小鼠脾淋巴细胞不分泌特异性IFN-γ;在DNA核酸疫苗免疫的四组小鼠脾淋巴细胞中,仅UbPrP组的淋巴细胞两只小鼠淋巴细胞能产生明显的斑点,分别为56、50 SFCs/106/PBMC,其他组的脾淋巴细胞在重组蛋白的刺激下均为阴性。小鼠抗血清ELISA结果显示,重组蛋白组免疫小鼠的抗血清的滴度为1∶51200,经质粒初免、重组蛋白增强小鼠抗血清效价在大于1∶102400,明显高于单纯重组蛋白免疫组;UbPrP组及PrPLⅡ组质粒免疫小鼠抗血清滴度大于1∶4 00;未修饰PrP组及空载体pcDNA3.1组小鼠抗血清与免疫前血清的P/N值小于2.1。各组抗血清特异性检测的WB结果显示:各组免疫小鼠的抗血清均可特异性识别重组人PrP。结论:成功构建了溶酶体、泛素定位表达的PRNP核酸疫苗载体pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ;与泛素融合表达及定位于溶酶体的PrP能一定程度地打破免疫耐受,诱导特异性免疫反应。更多还原

【Abstract】 Objective: To evaluate PrP expression characteristic and the basic data of PRNP nucleic acid vaccine with ubiquitin or the lysosome-targeting signal.Method: The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector, that is, pcDNA3.1-UbPrP and pcDNA3.1-PrPLⅡwere constructed. The expression characteristics of PrP with two signals were evaluated by western blot and the localization was observed by indirect immune fluorescence. The first four group mice were immuned by DNA vaccine. Each group was made of four mice, and each mouse received three DNA immunise at 14-day intervals. The other four group mice were immuned by DNA vaccine and boosted by recombinant prion protein. The immune responses of different groups were analyzed in 2 weeks after the last immunization. Specificity and sensitivity of antibodies were detected by both ELISA and Western blotting. Lymphocyte were collected and stimulated with recombinant human PrP protein from different groups, IFN-r was detected by ELISPOT.Result: The protein expressed by pcDNA3.1-UbPrP and pcDNA3.1-PrPLⅡwith ubiquitin and lysosome-targeting signal can be recognized by prion-specific antibody. The protein has three glycosylation molecules form as native PrP. PrP with ubiquitin was degraded gradually with time extension, whereas quantity of PrP with lysosome signal reduced in 48h after transfection. The protein with two location signals can direct fusion prion proteins into cytoplasm. The ELISPOT result show, there are 56、50 SFCs/106/PBMC in two mice of the UbPrP group respectively, 215、50SFCs/106/PBMCs in two mice of the UbPrP+rPrP group respectively, 113.5 SFCs/106/PBMCs in one mouse of the PrP+rPrP group. Lymphocyte from the other mice do not secreated PrP-specific IFN-γ. The ELISA results found that the titer of serum from mice immunized with a plasmid encoding both ubiquitin-tagged and lysosome-targeting signal PrP were above 1:400. But P/N levels of the serum titers from mice immunized with pcDNA3.1-PrP or pcDNA3.1 were below 2.1 The titer of serum from the protein boost group were much higher than the DNA immune group. The titers were above 1:51200 in the rPrP group, and the titers of mice with plasmid Primary Immunizied and protein boosted were above 1:102400. Antibodies induced after immunized with pcDNA3.1-UbPrP, pcDNA3.1-PrPLⅡ, pcDNA3.1-UbPrP+rPrP, pcDNA3.1-PrP+rPrP, pcDNA3.1-PrPLⅡ+rPrP could recognize the recombinant PrP proteinby Western bloting. The pcDNA3.1-UbPrP can breaked immune tolerance and enhanced both humoral and cellular responses against human prion protein.Conclusion: The PRNP vaccine with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. The vaccine with ubiquitin can break immune tolerance against the prion protein in wild-type mice, resulting in the induction of PrP-specific antibody and T-cell responses.更多还原