重组rPA基因在毕赤酵母细胞中的胞内表达研究

Intracellular expression of rPA gene in Pichia pastoris

目的构建含rPA全长基因的重组酵母胞内表达质粒pPIC9K rPA ,并在毕赤酵母中进行表达。方法用限制性内切酶BamHI和NotI将含有rPA全长基因的质粒pJZ1 6双酶切后 ,克隆至酵母表达载体pPIC9K中 ,构建酵母重组表达质粒。rPA基因经DNA序列测定准确无误。通过电穿孔法转染毕赤酵母菌GS1 1 5 ,PCR鉴定阳性酵母转化子 ,将rPA基因整合入酵母基因组DNA ,在毕赤酵母中实现了胞内非融合表达。表达产物进行SDS PAGE、Western blots以及溶纤维蛋白活性检测。结果表达产物以胞内包涵体的形式存在 ,未糖基化。SDS PAGE结果显示表达蛋白质的相对分子量约为 39kD。Western blots检测表明表达蛋白质能与单克隆抗体发生特异性反应。包涵体经 8mol L脲溶液溶解后 ,有明显的溶纤维蛋白活性。

Purpose To construct a recombinant plasmid pPIC9k rPA for the expression of whole length of rPA gene in Pichia pastoris. MethodsThe plasmid pJZ16 containing the whole length of rPA gene was digested with Bam H Ⅰ and Not Ⅰ and cloned into expression vector pPIC9k. Identified by sequence detemination and then transformed by electroporating.The positive transformants with the integrated rPA gene were identified by PCR and expressed in Pichia pastoris yeast cells. The expressed products were analyzed by SDS PAGE, Western blots and fibrin dissolving activity tests.ResultsThe expression of intracellular, insoluble, unglycosylated rPA was obtained. SDS PAGE showed a relative molecular weight of expressed protein of about 39 000 and Western blots showed the specific reaction of it with McAb. When dissolved in 8M urea, the inclusion body rPA exhibited fibrin dissolving activity.ConclusionThe recombinant rPA protein was successfully expressed in Pichia pastoris .