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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。
The apolipoprotein ApoE gene was amplified from the human cell line SH-SY5Y cDNA by RT-PCR and the fusion protein His-ApoE was expressed in the pET32a vector. The purified fusion protein was used to immunize rabbits to obtain specific antibodies, The interaction between ApoE and PrP was studied by ELISA and co-immunoprecipitation. The results showed that the relative molecular weight of His-ApoE protein expressed by SDS-PAGE was about 54 000, and the titer of the prepared antiserum was 1:406 900, which could effectively recognize the endogenous ApoE protein. ELISA and co-immunoprecipitation experiments showed that the prokaryotic expression of ApoE with the full-length PrP protein (PrP23-231) in vitro interaction occurs, the role of the site may be located in the N-terminal PrP protein. These results provide a new way to study the mechanism of TSE transmission through blood.