多种缺损及突变的PrP蛋白在杆状病毒中的表达和纯化  

Expression and Purification of Various Deleted and Mutated Hamster′s PrPs in a Baculovirus System

作　　者：张瑾[1] 张福萍[1] 聂凯[1] 陈岚[1] 周伟[1] 刘勇[2] 董小平[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所 [2]西安交通大学医学院,西安710061

出　　处：《病毒学报》2003年第1期36-41,共6页Chinese Journal of Virology

基　　金：国家自然科学基金委海外杰出青年项目 ( 3992 80 18);国家自然科学基金委项目 ( 30 0 70 0 38);国家自然科学基金委重点项目 ( 30 130 0 70 );国家 86 3计划项目 ( 2 0 0 1AA2 15 391)资助

摘　　要：PrP蛋白多肽序列上存在有不同的功能区域 ,影响着蛋白质构象、理化特性和生物学功能。为了获得不同缺损及突变的仓鼠PrP蛋白 ,以PCR方法获得不同大小的PrP基因片段 ,利用PCR大引物点诱变法获得带有突变糖基化位点PrP序列。所有PCR产物测序鉴定正确后 ,分别与pFASTBAC Hb质粒连接 ,与穿梭载体DH10BAC转座 ,构建各种重组病毒。Westernblot证实 ,8种不同长度的仓鼠PrP蛋白可在昆虫细胞Sf9中表达 ,包括全序列的PrP1 2 31,信号肽缺失的PrP2 3 2 31;N端缺损的PrP90 2 31和PrP12 0 2 31;C端缺损的PrP1 199、PrP1 174和PrP1 16 0。糖基化位点突变 :PrP2 3 2 31(N181Q)。其中不带信号肽序列的PrP蛋白均呈HIS融合蛋白 ,而N端带有信号肽的PrP蛋白则不能与Ni NTA琼脂糖亲和层析柱结合 ,但可以同PrP特异性抗体发生免疫沉淀反应。这提示PrP信号肽序列在昆虫细胞中可能也发挥着与哺乳动物细胞相似的作用。不同缺损及突变的PrP蛋白的表达和纯化为进行蛋白生物学性状研究奠定了基础。Various functional domains in PrP peptide have been reported,which influence the conformational,biological and biochemical characteristics of PrP protein.To study the potential functions of different regions of PrP peptide,various length fragments of hamster′s PrP were generated by PCR.In addition,a point mutation involved in a glycosylation site,leading to exchange of Asn to Gly at the position 181 in PrP peptide,was created by a mega-primer site-direct mutation PCR.After verified by sequence analyses,the PCR products were cloned into the vector pF ASTB ACHb and transposed with DH10BAC,generating respective recombinant baculoviruses.Eight PrP proteins of different length were expressed in transfected insect cell line Sf9 confirmed by Western blot assays,including the full-length PrP1-231,signal peptide deleted PrP 23-231,N-terminal deleted PrP90-231 and PrP120-231,glycosylation site mutated PrP23-231 (N181Q),C-terminal deleted PrP1-199,PrP1-174 and PrP1-160.All the signal peptide deleted PrP peptides were expressed in HIS fusion form that could be purified with Ni-NTA Agarose.Whereas,the PrP proteins with the signal peptide failed to absorb with Ni-NTA Agarose chromatograph,but could be immunoprecipitated with a PrP specific monoclonal antibody 3F4,indicating that the signal peptide of PrP might work similar in insect cells as in mammalian cells.This work lays good basis for further functional and conformational studies.

关 键 词：PRP 杆状病毒 基因表达 信号肽 

分 类 号：R373[医药卫生—病原生物学] Q78[医药卫生—基础医学]