人Lipocalin型前列腺素D合成酶真核载体的构建及酵母表达菌株的建立

Cloning and Eukaryotic Expression of Human Lipocalin-type Prostaglandin D Synthase in Pichia Pastoris

目的 :在毕赤酵母中表达人睾丸Lipocalin型前列腺素D合成酶 (L PGDS) ,以利于生物学功能及临床应用研究。 方法 :用PCR的方法从质粒pGEX 2T htL PGDS上扩增出人睾丸L PGDS基因编码序列 ,构建该基因的酵母表达质粒 ;将表达质粒电转化毕赤酵母 ,甲醇诱导L PGDS的表达。 结果 :PCR扩增的L PGDS成熟肽基因编码序列克隆T载体后 ,经测序证明与所报道的人睾丸L PGDScDNA完全一致。对培养上清进行SDS PAGE分析显示 ,在相对分子质量为 2 70 0 0处有重组蛋白的表达 ,与理论值完全一致。 结论 :人Lipocalin型前列腺素D合成酶在毕赤酵母中获得了高效。

Objectives: To express human testis Lipocalin type prostaglandin D synthase in Pichia Pastoris for further research on biological function and clinical applications. Methods: Human testis L PGDS gene coding region was amplified from plasmid pGEX 2T/htL PGDS by PCR with a deletion of the signal peptide sequence. The DNA fragment was inserted into pPIC9 to construct yeast expression plasmid followed by transformation of the yeast GS115 strain with electroporation. The recombinant his tag protein was induced to express by methanol. Results: The sequence of the amplified DNA fragment was identical to that of human testis L PGDS previously reported. The recombinant protein was found with a molecular mass of 27 000 on SDS PAGE, which was identical to that of native L PGDS. Conclusions: Secretory expression of human L PGDS was obtained in Pichia Pastoris .