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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1×10~6 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10–15μg/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10μg/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。
The expression and immunogenicity of Japanese encephalitis virus (JEV) prME protein were secreted and expressed by Pichia pastoris in order to lay the foundation for the development of JEV subunit vaccine. The prME gene of JEV SA14-14-2 strain was amplified by RT-PCR and ligated into the Pichia yeast expression vector pPICZa-A to obtain the pPICZa-prME plasmid and the pPICZa-SprME plasmid carrying the 19 Aa signal peptide at the C terminal of the JEV Cap protein . The expression vector was linearized with PmeI digestion, transformed into Pichia pastoris X33 by electroporation, and induced to undergo fermentation. SDS-PAGE and Western blotting were used to identify the expression of the target protein in the yeast fermentation supernatant. Purify the target protein by GE protein purification column, observe the target protein before and after purification by electron microscope, mix different concentrations of purified prME protein with Freund’s adjuvant and quantitatively purify the prME protein with different doses of nucleic acid adjuvant Four-week-old mice were immunized and blood was collected periodically. Antibody levels of serum from mice were detected by ELISA. Antibody neutralization titers were determined by plaque reduction assay. The result of SDS-PAGE showed that Pichia pastoris secreted the full-length prME protein and its target protein was between 70-100 kDa. The result of Western blotting showed that prME protein secreted by E.coli had good reactionality, which further proved that prME protein was expressed in yeast X33 Secreted expression as a whole without hydrolytic cleavage. Purification of the target protein, according to the elution time and volume showed that its molecular weight greater than 1 × 10 ~ 6 Da, thus infer prME protein may form multi-particle. Electron microscopy revealed 30-50 nm diameter virus like particles (VLPs). Immunoassay results showed that the purified recombinant protein 10-15μg / inoculated mice reached peak after 3 weeks, and then decreased gradually. After 7 weeks of immunization, JEV antibody could still be detected in the serum of mice. Mice were inoculated with prME VLPs at a dose of 10μg / dose with different doses of nucleic acid adjuvant. The results of ELISA showed that the nucleic acid adjuvant could significantly enhance the immune response of JEV prME VLPs. After 4 weeks of immunization, the neutralizing antibody The price is 1:80-1: 160. The above results indicate that, although Pichia pastoris-expressed JEV prME does not undergo hydrolytic cleavage, it still forms VLPs and induces immunized mice to produce higher levels of neutralizing antibodies.