定位表达于溶酶体、泛素的PrP载体的构建及鉴定  

The construction and identification of the PRNP vectors with ubiquitin or the lysosome-targeting signal

作　　者：李媛[1] 梅国勇[1] 姜慧英[2] 王桂荣[2] 田婵[2] 陈操[2] 王新[2] 王克霞[1] 韩俊[2] 董小平[2] 

机构地区：[1]安徽理工大学医学院病原生物学与免疫学教研室,淮南232001 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室

出　　处：《中华实验和临床病毒学杂志》2008年第6期419-421,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金委项目资助（No.30771914、30571672和30500018）;“863”计划（2006BAD06A13-2）;“973”计划（2007CB310505）.

摘　　要：目的构建定位于溶酶体、泛素的PrP表达载体，并进行蛋白表达特点及定位的鉴定。方法将泛素基因、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。结果构建的各种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、溶酶体信号的质粒pcDNA3．1-UPrP、pcDNA3．1-PrPL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PrP在细胞内的降解。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-uPrP、pcDNA3．1-PrPL，为PRNP核酸疫苗的研究奠定了一定的基础。Objective To evaluate PrP expression characteristic of PRNP nucleic acid vaccine vector with ubiquitin or the lysosome-targeting signal. Methods The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector that is, pcDNA3.1-UPrP and peDNA3.1-PrPL were constructed. The expression characteristics of PrP with two signals were evaluated by Westem Blot and the localization was observed by indirect immune fluorescence. Results The protein expressed by pcDNA3.1-UPrP and pcDNA3.1-PrPL with ubiquitin and lysosome-targeting signal can be recognized by prion-specifie 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 48 h after transfeetion. The protein with two location signals can direct fusion proteins to cytoplasm. Conclusion The PRNP vectors with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. There will be one of good foundation on PRNP nucleic acid vaccine.

关 键 词：朊病毒 定位表达 疫苗 DNA 

分 类 号：R346[医药卫生—基础医学]