大菱鲆红体病虹彩病毒主要衣壳蛋白基因在毕赤酵母中的重组分泌表达

依据大菱鲆红体病虹彩病毒（Turbot viral reddish body iridovirus，TRBIV）主要衣壳蛋白（Major capsid protein，MCP）基因序列，设计了一对特异性引物，并在正反向引物中分别引入EcoR Ⅰ和Not Ⅰ酶切位点，从而将PCR扩增得到的TRBIV MCP基因双酶切后定向克隆到真核表达载体pGAPZαA的GAP启动子的下游位点，并电转化入大肠杆菌DH5α宿主菌内。经抗生素Zeocin筛选、PCR、EcoR Ⅰ和Not Ⅰ双酶切以及测序分析，构建了含有α-factor信号肽的真核重组表达载体pGAPZαA MCP。重组表达质粒pGAPZαA MCP经Avr Ⅱ单酶切后电转导入毕赤酵母X-33中，挑选阳性克隆，提取表达上清经SDS-PAGE和Western-blot免疫印迹分析。结果显示，TRBIV MCP基因在酵母中成功实现分泌表达。阳性重组酵母菌经过72h诱导培养后，重组TRBIV MCP的表达量高达60.2μg/ml左右。

Expression of major capsid protein gene of turbot reddish body iridovirus in Pichia pastoris

A pair of PCR primers, with EcoR Ⅰ and Not Ⅰ restricted enzyme sites in the 5＇ and 3＇ end respectively, was designed according to the major capsid protein （MCP） gene of turbot viral reddish body iridovirus （TRBIV）. After PCR amplification, a recombinant expression vector including α-factor peptide and 6 × His tag was constructed by inserting TRBIV MCP gene into GAP promoter downstream of pGAPZαA directly. The constructed vector pGAPZαA MCP was linearized by Avr Ⅱ , and then transformed and intergrated into the host Pichia pastoris X-33 genome by electroporation. The recombinant yeasts with high-level secreted expression of MCP were screened by ELISA-Dot. The expressed proteins were identified by SDS-PAGE and Western blot. The results revealed that a recombinant protein with the molecular weight of approximately 51kDa was secreted into the supernatant from the recombinant yeast cells successfully. The expressional amount of TRBIV MCP could reach to 60.2 μg/ml in the supernatant secreted from recombinant yeast after being fermented at 28 ℃ for 72 h.