不同接头的白蛋白干扰素在毕赤酵母中表达的研究

Study on Pichia. Pastorof expression for HSA-IFN α-2b of different linker

毕赤酵母作为比较成熟的真核表达系统[1],它具有许多优点,表达产物直接分泌到胞外,这有利于产物的分离纯化。白蛋白干扰素在毕赤酵母中表达的研究也具有很重要的意义。研究白蛋白与干扰素之间的接头蛋白不同对表达量的影响。通过重叠PCR构建不同接头的白蛋白干扰素重组子,然后分别用salⅠ酶切线性化,通过电转仪转入毕赤酵母SMD1168,在MD板上进行培养,分别挑取10个克隆进行甲醇诱导表达,筛选出表达量最高的一个。最后在相同的条件下,分别将表达量高的进行诱导表达,表达产物通过SDS-PAGE鉴定,由于表达产物中有杂蛋白,通过酶联免疫吸附法(ELISA)测定融合白蛋白干扰素的表达量,由已知浓度的白蛋白干扰素标准曲线,可以计算出不同接头的白蛋白干扰素的表达量,这对工业化生产具有指导意义。

As a useful eukaryotic expressing system, Pichia pastoris has many advantages, products of expressing directly secreted into the extracellular, which is conducive to the purification of product. Albumin interferon in Pichia pastoris expression also has very important significance. This experiment studied the connection between albumin and interferon of different linker on the protein of expression. Different linker recombinant interferon albumin was constructed by overlapping PCR, and then lineafized with sal I digested and electroporation into SMDl168 of Pichia pastoris, cultured in the MD board, 10 clones were picked by methanol induction, selected one of the highest. The protein expression products were detemined in a complex by enzyme-linked immunosorbent assay （ELISA） and calculated for different linker expression of albumin interferon.