毕赤酵母分泌表达JEV prME蛋白及其形成病毒样颗粒的免疫原性鉴定

Secreted expression of Japanese encephalitis virus prME in Pichia pastoris and immunogenicity evaluation of the virus-like particles in mice

摘要:

应用毕赤酵母分泌表达日本脑炎病毒 (Japanese encephalitis virus，JEV) prME蛋白，鉴定其表达效果与免疫原性，以期为JEV亚单位疫苗的研制奠定基础。RT-PCR扩增JEV SA14-14-2株 prME基因，将其连接到毕赤酵母表达载体pPICZa-A，分别获得pPICZa-prME和携带JEV Cap蛋白C末端19个Aa信号肽的pPICZa-SprME质粒。表达载体用PmeⅠ酶切线性化，通过电转化转入毕赤酵母X33并诱导发酵培养。利用SDS-PAGE和Western blotting鉴定酵母发酵上清中目的蛋白的表达情况。利用GE蛋白层析纯化柱纯化目的蛋白，利用电镜观察纯化前后的目的蛋白，将不同剂量纯化后的prME蛋白与弗氏佐剂混合以及定量纯化后的prME蛋白与不同剂量的核酸佐剂混合分别免疫4周龄小鼠，定期采血，ELISA检测被免小鼠血清的抗体水平，空斑减少试验测定抗体中和效价。SDS-PAGE结果表明毕赤酵母可以分泌表达完整的prME蛋白，目的蛋白在70–100 kDa之间；Western blotting结果显示分泌表达的prME蛋白具有良好的反应原性，进一步证明prME蛋白在酵母X33中以整体的形式分泌表达，没有发生水解切割。纯化目的蛋白，根据洗脱时间和体积表明其分子量大于1×106 Da，因此推断prME蛋白可能形成多聚化的颗粒。电镜观察发现直径30–50 nm的病毒样颗粒 (Virus like particles，VLPs)。免疫试验结果表明，纯化后的重组蛋白10–15 μg/只接种小鼠在3周后抗体达到高峰值，之后逐渐下降，免疫7周后小鼠血清仍可检测到JEV抗体。将prME VLPs以10 μg /只的剂量与不同剂量的核酸佐剂配伍后接种小鼠，ELISA检测结果表明核酸佐剂可明显增强JEV prME VLPs免疫应答，免疫4周后小鼠血清的中和抗体效价为1∶80–1∶160。上述结果表明毕赤酵母表达JEV prME虽不能发生水解切割，但仍可形成VLP并诱导免疫小鼠产生较高水平中和抗体。

Abstract:

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.