Zhao Peng, Jiang Ya, Wang Jingman, Fan Haojie, Cao Ruibing
Key Laboratory of Animal Bacteriology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2017 May 25;33(5):863-874. doi: 10.13345/j.cjb.170026.
The study was to express prME protein of Japanese encephalitis virus (JEV) in Pichia pastoris and then to evaluate the immunological properties of the recombinant protein in mice, so as to explore a new way for subunit vaccine development of JEV. The JEV prME gene was amplified by RT-PCR with genome RNA of JEV vaccine strain SA14-14-2 and subcloned into pPICZa-A vector, designated as pPICZα-prME. pPICZα-SprME was constructed same as pPICZα-prME besides with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal. The linearized expression vector was integrated into the genome of Pichia pastoris X33 under the control of the alcohol oxidase (AOX1) promoter and induced with methanol during fermentation expression. The expression of JEV prME protein was identified by SDS-PAGE and Western blotting, and then it was purified by S-400 High Resolution HiPrep 16/60 Sephacry. The expressed products of Pichia pastoris were visualized by electron microscopy. In the immunization test, four groups of four-week old female mice were immunized subcutaneously with different doses purified JEV prME protein with complete Freund's adjuvant at a volumetric ratio of 1:1 and a control group was injected with sterile PBS. 10 μg/dose purified JEV prME protein mixing different doses nucleic acid adjuvant (Naa) was vaccinated in mice as the same mode. SDS-PAGE and Western blotting indicate that JEV prME was not cleaved between prM and E during secreted expression in Pichia pastoris. The purified recombinant prME was eluted in the first eluting peak which indicated that its molecular weight about 1×10⁶ Da to 20×10⁶ Da and may form a multimeric. Both the culture supernatant and the purified protein, examined by electron microscopy, we found to contain JEV virus like particles (VLPs) with diameters of 30-50 nm. The anti-JEV VLPs antibody titration reached peak at 3 wpi and still maintained in mice at 7 wpi inoculated with 10 μg and 15 μg prME. The strong antibody response was observed when the mice immunized with prME mixing nucleic acid adjuvant, which elicited high neutralizing antibody titer among 1:80 to 1:160. In conclusion, although JEV prME protein expressed in Pichia pastoris was not cleaved, which formed VLPs and showed efficient immunological properties in mice experiments.
本研究旨在利用毕赤酵母表达日本脑炎病毒（JEV）的prME蛋白，进而评估该重组蛋白在小鼠体内的免疫特性，为JEV亚单位疫苗的研发探索新途径。以JEV疫苗株SA14 - 14 - 2的基因组RNA为模板，通过RT - PCR扩增JEV prME基因，并将其亚克隆至pPICZa - A载体，命名为pPICZα - prME。除了含有JEV衣壳蛋白C末端额外的19个氨基酸信号肽编码基因外，按照构建pPICZα - prME的方法构建pPICZα - SprME。将线性化的表达载体在醇氧化酶（AOX1）启动子的控制下整合到毕赤酵母X33的基因组中，并在发酵表达过程中用甲醇诱导。通过SDS - PAGE和Western印迹鉴定JEV prME蛋白的表达，然后用S - 400高分辨率HiPrep 16/60琼脂糖凝胶进行纯化。用电子显微镜观察毕赤酵母的表达产物。在免疫试验中，将四组四周龄雌性小鼠皮下注射不同剂量的纯化JEV prME蛋白与完全弗氏佐剂，体积比为1:1，对照组注射无菌PBS。以相同方式用10μg/剂量的纯化JEV prME蛋白混合不同剂量的核酸佐剂（Naa）对小鼠进行接种。SDS - PAGE和Western印迹表明，JEV prME在毕赤酵母分泌表达过程中在prM和E之间未被切割。纯化的重组prME在第一个洗脱峰中被洗脱，表明其分子量约为1×10⁶ Da至20×10⁶ Da，可能形成多聚体。通过电子显微镜检查，发现培养上清液和纯化蛋白均含有直径为30 - 50nm的JEV病毒样颗粒（VLP）。接种10μg和15μg prME的小鼠在接种后3周时抗JEV VLP抗体滴度达到峰值，在7周时仍保持较高水平。用prME混合核酸佐剂免疫小鼠时观察到强烈的抗体反应，诱导出1:80至1:160的高中和抗体滴度。总之，尽管在毕赤酵母中表达的JEV prME蛋白未被切割，但形成了VLP，并在小鼠实验中显示出有效的免疫特性。
