对虾白斑综合征病毒囊膜蛋白VP28和VP26的毕赤酵母组成型分泌表达

Secretive Expression of White Spot Syndrome Virus Envelope Proteins VP28 and VP26 in Pichia pastoris Induced by Constitutive Promoter

近年来,重组VP28和VP26蛋白作为蛋白亚单位疫苗,在增强对虾抗白斑综合征病毒(WSSV)感染的过程中具有重要作用。本研究根据Gen Bank中WSSV的基因序列设计引物,以WSSV粗提液为模板进行普通PCR扩增,得到VP28和VP26基因,再用引物悬挂法将Eco RⅠ和XbaⅠ酶切位点分别添加到VP28和VP26基因的5端和3端。目的基因经双酶切后插入到表达载体p GAPZαA,转化TOP10大肠杆菌,经博莱霉素(Zeocin)抗性筛选阳性重组酵母表达载体。AvrⅡ酶切线性化之后,电击转化X-33毕赤酵母感受态细胞,经Zeocin抗性筛选得到阳性重组酵母。SDS-PAGE电泳分析重组酵母表达上清液的目的蛋白,没有检测到VP28和VP26重组蛋白。随后,采用蛋白质银染法,结果显示,与空载p GAPZαA组相比,VP28和VP26表达上清液组有明显的条带,证明VP28和VP26在毕赤酵母中成功表达,蛋白分子量大小约为32 kDa。

WSSV has been a globally recognized highly harmful pathogen in shrimp farming industry that causes tremendous economic loss. The envelope proteins of WSSV, VP28 and VP26, play important roles in interacting with host cells, initiating virus infection and mediating virus intrusion. In this study, we used pGAPZαA as the expression vector and X-33Pichia pastoris as the host cell to express VP28 and VP26 in a secretive manner. The coding sequences of VP28 and VP26 （GenBank： AF332093.3） were amplified from WSSV using PCR, and the sequences ofEcoR I （GAATTC） andXba I （TCTAGA） were added to the 5￠ and 3￠ ends of the target genes. The purified PCR products were then cloned into theEcoR I/Xba I sites of the pGAPZαA vector. Sequencing analysis verified whether the target genes were correctly inserted into the reading frame. The construct was linearized byBln I （AvrⅡ） and then was integrated into P. pastoris X-33 through electroporation while being screened by Zeocin. The expressed proteins were identified with SDS-PAGE. The VP28 and VP26 recombinant proteins could not be detected by coomassie brilliant blue R250 staining, however, the bands of the fusion proteins appeared after silver staining. The sizes of VP28 and VP26 fusion proteins were about 32 kDa. These results suggest that theP. pastorissystem was effective in expressing WSSV envelope proteins VP28 and VP26, although the expression level was not sufficient. Nonetheless, our study still established a novel tool for the study of subunit vaccine, and provided basic information for the large scale vaccine production.