摘要
利用RT-PCR方法从人源细胞系SH-SY5Y cDNA中扩增出载脂蛋白ApoE基因,并在pET32a载体中表达融合蛋白His-ApoE,以纯化的融合蛋白免疫实验用兔获得特异性抗体,利用ELISA及免疫共沉淀方法对ApoE及PrP之间的相互作用进行研究。结果表明,SDS-PAGE显示表达的His-ApoE蛋白的相对分子量约为54 000,所制备的抗血清ELISA效价可达到1∶406 900,并能够有效地识别重组及动物组织中的内源性ApoE蛋白。ELISA和免疫共沉淀实验均显示,原核表达的ApoE可与全长的PrP蛋白(PrP23-231)在体外发生相互作用,其作用位点可能位于PrP蛋白的N端。这些结果为TSE经血传播的机制研究提供了新思路。
In order to study the possible molecular interaction between PrP protein and the prokaryotic-expressed apolipoprotein E, the full-length sequence of human ApoE gene was amplified with RT-PCR using the mRNA from cell line SH-SY5Y as the template, and inserted into a prokaryotic-expressing plasmid pET32a. The purified fusion protein was emulsified with complete or incomplete Freund＇ s adjuvant and injected subcutaneously into rabbits. The interaction between ApoE and PrP was evaluated with ELISA and immunoprecipitation assays. SDS-PAGE showed that the molecular weight of the expressed fusion protein HIS-ApoE was about 54000. ELISA assay revealed that the reacting titer of the prepared antiserum reached to 1 ： 406900, while Western blot confirmed that the prepared antiserum was able to recognize both the recombinant ApoE protein and the native ApoE from various mammalian＇ s liver tissues. Using special ApoE-coated ELISA and immunoprecipitation tests, the activities of molecular interaction between ApoE and PrP were detected. The binding domain lies within the N-terminus of PrP protein.
出处
《病毒学报》
CAS
CSCD
北大核心
2005年第6期443-447,共5页
Chinese Journal of Virology
基金
国家自然科学基金委项目(30070038)
国家自然科学基金委重点项目(30130070)
国家863计划项目(2001AA215391)
欧盟项目(QLRT200001441)
国家科技攻关计划项目(2003BA712A04-02)资助
