猪血凝性脑脊髓炎病毒HE基因的克隆及真核表达

Cloning and Eukaryotic Expressing of HE Gene of Hemagglutinating Encephalomyelitis Virus

应用特异性引物,从猪血凝性脑脊髓炎病毒(HEV)中扩增出HE蛋白基因,PCR产物纯化后克隆入pGEM-T载体,得到重组质粒pTHE。用EcoRⅠ和NotⅠ双酶切pTHE,回收目的基因HE片段并将其定向克隆到pPICZαA中,构建重组质粒pPICZαA HE。用BstXⅠ酶切pPICZαA HE使其线性化,并电击转化至感受态毕赤酵母细胞GS115。PCR法鉴定阳性重组子,用1%甲醇诱导表达后,进行SDS-PAGE及Western blot分析。结果在酵母菌培养基上清中检测到相对分子量为43 kD的重组蛋白,且该重组蛋白可与HEV多克隆抗体发生特异性血清学反应,表明HEV的HE蛋白片段在Pichia Pastoris中获得成功表达。

To clone and express HE protein of HEV in Pichia Pastoris yeast, HE gene was amplified by PCR from HEV with a pair of specific primers. Then PCR products were purified and cloned into pGEM- T vector to obtain the plasmid pTHE. The intersting gene fragment was recovered after the double enzyme digestion of Eco R Ⅰ and Not Ⅰ , then subcloned into pPiCZαA for secretory expression. The recombinant pPiCZαAHE was lineaized with BstX Ⅰ and then transformed into GS115 yeast cells by electmporation for expression under the induction of 1% methanol. The recombinated strains were screened by PCR technique. The expression products were identified by SDS-PAGE and western-blot. The results showed that there was a molecular weight of 43 kD, which could be specifically recognized by polyclonic antibody against HEV. It suggested that the HE protein of HEV was obtained with Pichia Pastoris expression system.