PrP(D178N)无细胞表达、抗原性及结构分析

Cell-free Expression,Antigenicity and Structure of PrP(D178N)

目的无细胞表达人178位点突变朊蛋白PrP(D178N),并分析其抗原性及结构。方法提取人血液基因组DNA,应用PCR法突变人PrP 178号位点。以正常的PrP为对照组,分别连接至真核表达载体pcDNA3.1上,利用无细胞真核表达系统进行表达。Western blot分析表达的重组蛋白的抗原性,电镜观察抗原结构。结果突变基因重组表达质粒构建正确;表达的PrP(D178N)蛋白相对分子质量约为28 000,对PK酶具有抗性,电镜下可见氧病相关纤维(Scrapie associated fibrils,SAF)存在。结论 已成功无细胞表达了PrP(D178N),为后续其突变后空间构象及致病机理的研究奠定了基础。

Objective To express human prion protein with mutation at site 178,i.e.PrP（D178N）,by cell-free expression technique,and analyze its antigenicity and structure.Methods Genomic DNA of human blood was extracted,to which the mutation at human PrP178 site was introduced by PCR.The PCR product was inserted into vector pcDNA3.1 for expression in cell-free eukaryotic expression system,using normal PrP as control.The expressed protein was analyzed for antigenicity by Western blot,and observed for antigen structure by electron microscopy.Results The recombinant plasmid with mutant gene was constructed correctly.The expressed PrP（D178N）,with a relative molecular mass of about 28 000,showed resistance to PK,and was scrapie associated fibrils（SAF）-positive under electron microscope.Conclusion PrP（D178N） was successfully expressed by using cell-free expression technique,which laid a foundation of further study on its optical confirmation and pathogenic mechanism.