利用GST融合蛋白表达系统表达人PrP蛋白

Expression of human prp gene in prokaryotic cells using GST fusion protein expression system

目的研究与人类海绵状脑病发病有关的膜蛋白ＰｒＰｃ的生物学功用，同时以其为抗原建立有效的免疫学诊断方法。方法采用ＰＣＲ扩增并克隆２例中国人ｐｒｐ基因，经ＤＮＡ序列分析后，亚克隆至ＧＳＴ融合蛋白表达质粒。结果１例正常人ｐｒｐ基因带有点突变，导致形成终止密码，另１例ｐｒｐ基因序列与已发表的标准ｐｒｐ基因序列相同。在ＧＳＴ融合蛋白表达系统中，标准及突变ｐｒｐ基因可有效地表达完整及缺损的ＰｒＰ蛋白。Ｗｅｓｔｅｒｎ蛋白转印试验证实，所表达的ＧＳＴＰｒＰ融合蛋白具有良好的免疫反应性。结论人ｐｒｐ基因能在大肠杆菌ＧＳＴ融合蛋白表达系统中得到高效地表达。

Objective To study the biological features of cellsurface protein PrPc, which is thought to be involved in the prionassociated diseases after converting to a proteinaseresistant isoform PrPSc posttranslationally, and to establish an effective immunologic diagnostic method using PrPc as antigen. Methods Amplifying and cloning the human prp gene from lymphocytes of two normal Chinese, after confirmed by DNA sequence analysis, the genes were separately subcloned into a GSTfusion expression plasmid. Results Sequence analysis showed that one contatined a point mutation that induced the 65th amino acid Trp inverting to a stop codon TAG, whereas the other had the same sequence as the published standard prp gene.Both the standard and the mutated prp genes were separately subcloned into a GST fusion protein expression vector and expressed in the prokaryotic cells effectively. Western blot assay revealed that both of them expressed GSTPrP fusion proteins and could be recognized by PrP specific monoclonal antibody. Conclusion It suggests that human PrP protein can be expressed in the GST fusion protein expression system and the expressed proteins hold good immunereactivity.