猪圆环病毒3型Cap蛋白的高密度发酵工艺及免疫评价  

High density fermentation and immune evaluation of porcine circovirus type 3Cap protein

作　　者：李可乐 许景龙 耿笑林 王志飞 梁严予 莫晓琳 黄玉欣 逄文强 田克恭 LI Kele;XU Jinglong;GENG Xiaolin;WANG Zhifei;LIANG Yanyu;MO Xiaolin;HUANG Yuxin;PANG Wenqiang;TIAN Kegong(National Research Center for VeterinaryMedicine,Luoyang,Henan 471003,China;Pulike Bioengineering Co.,Ltd.,Luoyang,Henan 471003,China)

机构地区：[1]国家兽用药品工程技术研究中心,河南洛阳471003 [2]普莱柯生物工程股份有限公司,河南洛阳471003

出　　处：《中国兽医学报》2024年第12期2507-2513,共7页Chinese Journal of Veterinary Science

基　　金：“十四五”国家重点研发计划资助项目(2021YFD1801302-2,2023YFD1800505)。

摘　　要：为了实现猪圆环病毒3型(PCV3)Cap蛋白在重组大肠杆菌中的高效表达,在10L发酵罐中考察溶氧(DO)控制策略、氧消耗速率(OUR)控制策略、DO和OUR联合控制策略对目的蛋白表达的影响;通过选择最佳的OUR控制范围确定重组大肠杆菌的高密度发酵工艺;对高密度发酵的蛋白进行SDS-PAGE、Western blot鉴定和免疫评价。结果表明:DO控制策略和OUR控制策略的目的蛋白表达量偏低,检测发现乙酸是影响目的蛋白表达量的主要因素,当乙酸质量浓度达到1.02g/L时目的蛋白的表达量降低55.8%。DO和OUR联合控制策略在补料前以DO作为控制供氧的参数,在补料后以OUR作为控制供氧的参数,保持OUR值在每小时140~160mmol/L,可以减少乙酸的生成,目的蛋白的表达量最高为650mg/L。Western blot鉴定结果显示目的蛋白具有良好的反应原性和特异性。动物试验结果显示二免后抗体阳性率为100%,目的蛋白具有良好的免疫原性。说明DO和OUR联合控制的高密度发酵工艺可以实现PCV3Cap蛋白在发酵罐上的高效可溶性表达,为进一步进行疫苗研制奠定了基础。In order to achieve high-efficiency expression of the porcine circovirus type 3Cap protein in recombinant Escherichia coli,dissolved oxygen(DO)control strategy,oxygen uptake rate(OUR)control strategy and combined control strategy of DO and OUR were selected to investigate their effect on the expression of target protein in a 10Lfermenter.The high-density fermentation process of recombinant E.coli was determined by investigating the best control range of OUR.The recombinant protein was identified by SDS-PAGE and Western blot,and the immunogenicity of the protein was evaluated by the animal experiment.The results showed that when the DO control strategy and OUR control strategy were used,the expression of target protein was low.Acetic acid was the key factor affecting the expression of target protein.When acetic acid concentration reached 1.02g/L,Cap protein yield was reduced by 55.8%.In order to reduce the production of acetic acid,DO was selected to control oxygen supply before feeding,and OUR was selected as the parameter to control oxygen supply after feeding.When OUR value was maintained at 140-160mmol/(L·h),the Cap protein yield was up to 650mg/L.Western blot analysis confirmed that PCV3Cap possessed antigenicity and specificity.Animal experiment showed that the antibody positive rate was 100%after the second immunization,indicating that the target protein had better immunogenicity.The high density fermentation controlled by the combined control strategy of DO and OUR could achieve efficiently soluble expression of PCV3Cap in the fermenter,which laid the foundation for the vaccine development.

关 键 词：PCV3Cap蛋白 乙酸 DO和OUR联合控制 高密度发酵 

分 类 号：S852.65[农业科学—基础兽医学]