猪圆环病毒2d型病毒样颗粒疫苗的制备及发酵工艺研究

Study on preparation and fermentation technology of porcine circovirus 2d virus-like particles vaccine

为制备猪圆环病毒2d型(PCV2d)病毒样颗粒(VLPs)疫苗并对其免疫效果进行评价。本研究采用原核表达系统表达PCV2d Cap蛋白,并对其发酵工艺条件进行了优化,去除His-SUMO标签使其体外自组装形成VLPs,通过SDS-PAGE和Western-blot进行验证,采用透射电镜和纳米粒度仪进行表征,将验证正确的VLPs免疫小鼠,测定其抗体水平和细胞因子水平。结果显示,在0.5 mmol/L IPTG、N源补料速率为12%时,蛋白表达量最高,利用透射电镜、纳米粒度仪进行表征鉴定,发现PCV2d可自组装形成VLPs,粒子直径约为20~25 nm。PCV2d VLPs与ISA 201佐剂混合免疫小鼠,特异性抗体滴度达到1∶512以上,中和抗体滴度达到1∶32以上,IL-4和IFN-γ细胞因子水平均可达到80 pg/m L,且明显高于PBS组。综上所述,本研究利用原核表达系统构建表达并成功获得PCV2d VLPs,且可以诱导机体产生良好的细胞免疫和体液免疫,这为猪圆环病毒2d型疫苗及猪圆环病毒多价疫苗的研制奠定了基础。

To develop a porcine circovirus type 2d virus-like particles vaccine and evaluate its immunization effect,the PCv2d Cap protein were expressed by prokaryotic expression system and confirmed through SDS-PAGE and Western-blot analysis.When the prime conditions of fermentation process were screened,the His-SUMO tag of expressed PCv2d Cap was removed to make its self-assembly into VLPs in vitro.The morphological characterization of the VLPs was checked by transmission electron microscopy and nanoparticle-size analyzer.The antibody and cytokine levels in sera were detected at 28 d after mice immunized with the VLPs.The results showed that the highest level of protein expression was achieved at 0.5 mmol/L IPTG and a 12%N source feed rate,and the VLPs were found to have a particle diameter of approximately 20-25 nm.The titers of specific antibody and neutralizing antibody were more than 1:512 and 1:32 respectively,and the levels of IL-4 and IFN-γ cytokines were 80 pg/mL,which were significantly higher than those in the PBS group.In conclusion,this study successfully constructed and obtained PCv2d VLPs using the prokaryotic expression system,which induced good cellular and humoral immunity and laid the groundwork for research on the porcine circovirus 2d subtype and porcine circovirus multivalent vaccine.