水牛朊蛋白成熟片段基因的克隆与原核表达

【目的】克隆、分析水牛朊蛋白（prion protein，PrP）成熟片段基因，并获取纯化的表达产物。【方法】将应用PCR技术扩增的水牛成熟PrP的核酸序列克隆到表达载体pET-32a，并分析其序列；把阳性克隆质粒pET-32a-BPrP转化表达菌BL21(DE3)，鉴定纯化的表达产物。【结果】水牛朊蛋白成熟片段核酸序列及其推导的氨基酸序列与已知其他牛科动物的成熟PrP核酸、氨基酸序列间的相似性分别在97.1%和97.2%以上，并且发现水牛成熟PrP有5个重复序列，编码的氨基酸序列其中有2个九肽和3个八肽重复序列。具有较高的表达水平的PrP融合蛋白被纯化后，用Western印迹实验证明该蛋白具有PrP抗原活性。【结论】首次报道了克隆、分析水牛朊蛋白成熟片段基因，发现水牛成熟PrP有5个重复序列，并获得了具PrP抗原活性的水牛成熟PrP融合蛋白。

Cloning, Prokaryotic Expression of the Buffalo Mature PrP Gene

Objective The study was conducted in order to clone and analyze the buffalo mature PrP gene and to acquire the purified recombined PrP protein.MethodWith the polymerase chain reaction (PCR) method, the DNA sequence encoding the buffalo mature PrP (Prion Protein) was amplified. It was then cloned into the expressing vector pET-32a. It was sequenced and analyzed. Positive recombinant plasmids pET-32a- BPrP to BL21 (DE3) were then transformed.Lastly, the expressing products would be examined. ResultAmong the documented PrP sequences of the family Bovidae and the PrP sequence reported here, the homology analysis results showed the similarities of nucleotides and proteins among them were above 97.1% and 97.2%,. The buffalo mature PrP gene contains five copies of a short, G-C-rich element that encodes the octapeptide,. There are two nonapeptides and three octapeptides. The recombined PrP protein was produced at a high level by BL21 (DE3) and it was purified. The result indicated it has the antigenicity of PrP in the Western blotting test.ConclusionThis is the first report about cloning and analyzing the buffalo mature PrP gene. There are five repeat sequences in the gene. And the purified recombined PrP protein was verified has the antigenicity of PrP.