猪圆环病毒2型（PCV2）是引起断奶猪多系统衰竭综合症（PMWS）的主要致病原，在全世界范围内对养猪业造成了巨大的经济损失。猪圆环病毒2型Cap蛋白（PCV2-Cap）是制备猪蓝耳病疫苗的主要靶蛋白。针对目前PCV2疫苗生产工艺复杂、成本较高的问题，本研究利用毕赤酵母表达系统研究PCV2-Cap蛋白的重组表达及疫苗活性，将PCV2-Cap基因序列去除核定位信号序列（NLS）后，经密码子偏好性优化，构建重组表达载体，并转化至毕赤酵母宿主菌X-33，经Zeocin抗性筛选获得重组菌株。SDS-PAGE和Western blot结果显示Cap蛋白在酵母表达系统中成功表达，蛋白质大小为26 ku。5 L罐高密度发酵小试，得到PCV2-Cap蛋白在发酵上清液中的浓度约为0.53 mg/mL，占总蛋白的23.5%。动物免疫试验显示重组Cap蛋白能刺激机体产生特异性抗体。结果表明，PCV2-Cap基因在毕赤酵母中实现了分泌表达，为PCV2-Cap毕赤酵母亚单位疫苗的研制提供了数据支持。

Porcine circovirus type2 (PCV2) is the major pathogen of weaning multi-systemic wasting syndrome (PMWS), which has caused significant economic losses to the swine industry worldwide. Porcine circovirus type 2 capsid protein（PCV2-Cap）is the main target protein for the preparation of vaccine against weaning multi-systemic wasting syndrome (PMWS). In this study, the recombinant expression and vaccine activity of PCV2-Cap protein in Pichia pastoris were investigated to solve the problems of complex production process and high cost of PCV2 vaccine. After removing the nuclear localization sequence (NLS) from PCV2-Cap gene sequence, a preferential optimization of codon was performed to construct a recombinant expression vector and transformed into the host bacteria (X-33) of P. pastoris. The results of SDS-PAGE and Western blot showed that the Cap protein was successfully expressed in the yeast expression system with a protein size of 26 Ku, and the high density fermentation test in 5 L bioreactor suggested that the concentration of PCV2-Cap protein in fermentation supernatant was 0.53 mg/mL, accounting for 23.5% of the total cell protein. In addition, the animal immunization test demonstrated that Cap protein could stimulate the body to produce specific antibodies. Consequently, the PCV2-Cap gene was secreted and expressed P. pastoris, which might make a contribution to the development of PCV2-Cap subunit vaccines.

广东省科技计划项目（2016A050503016、2016A010105004）；广州市科技计划项目（201510010191）；广东省自然科学基金项目（2017A030313097）