含EB病毒LMP2A毕氏酵母表达载体的构建与表达LMP2A蛋白的检测

Expression of LMP2A Gene of Epstein-Barr Virus in Pichia pastoris

目的:构建EB病毒潜伏膜蛋白2A的表达载体,并在真核生物毕氏酵母细胞中表达。方法:用EcoR I和Not I将含LMP2A全长编码基因的质粒PCIcc双酶切后,克隆到毕氏酵母载体PICZαA中,构建重组表达质粒pPICZαA-LMP2A。pICZαA-LMP2A经Sac I酶切线性化,采用氯化锂的方法转化毕氏酵母GS115,经YPD平板Zeocine抗性筛选获得LMP2A阳性克隆的菌株,用PCR鉴定阳性酵母转化子,并分别将阳性的酵母转化子在BMGY和BMMY培养基中进行诱导表达,表达产物进行SDS-PAGE电泳和western-blotting分析。结果:重组质粒pPICZαA-LMP2A经PCR和酶切鉴定为阳性。SDS-PAGE电泳和western-blotting结果显示表达蛋白的相对分子量为54 Kda,与预计值相同。结论:构建了LMP2A毕氏酵母表达载体,并在酵母细胞中成功表达。

Objective: To construct a recombinant plasmid pPICZαA-LMP2A for the expression of latent-infection membrane protein 2A(LMP2A) gene in Pichia pastoris. Methods: The plasmid pPCIcc containing the full length of LMP2A cDNA was digested with EcoR I and Not I and cloned into expression vector pPICZαA. The constructed plasmid pPICZαA-LMP2A was linarized with Sac I and transformed to Pichia pastoris GS115. The transformants were selected by planting cells on YPD/Zeocine plates, and then identified by PCR and expressed in BMGY and BMMY media. The expression product was analyzed by SDS-PAGE. Results: Based on the result of the 12% SDS-PAGE and western-blot, a recombinant protein about 53 KDa expressed in the yeast supernatant was identified to be recombinant LMP2A. Conclusion: The LMP2A cDNA successful expression was the foundation of further research in its fuction and constructed the suitable genetic engineering vaccine against EBV associated disease.