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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 PrP (D178N), a human 178-site mutant, and analyze its antigenicity and structure. Methods Human blood genomic DNA was extracted and the human PrP 178 site was mutated by PCR. The normal PrP as control group, respectively, were linked to the eukaryotic expression vector pcDNA3.1, the use of cell-free eukaryotic expression system for expression. Western blot analysis of the expression of recombinant protein antigenicity, electron microscopy antigen structure. Results The recombinant plasmid was constructed correctly. The relative molecular mass of expressed PrP (D178N) protein was about 28 000, which was resistant to PK enzyme. Scratch of associated fibrils (SAF) was observed under electron microscope. Conclusion PrP (D178N) has been successfully expressed in cell-free manner, which lays the foundation for the subsequent research on the spatial conformation and pathogenesis of the mutation.