表达猪瘟病毒E2蛋白的重组杆状病毒反应器工艺研究  

Improvement of Bioreactor Process of Recombinant Baculovirus Expressing E2 Protein of Classical Swine Fever Virus

作　　者：陈晓 李伟国 宋欢欢 苏晓蕊 王同燕 谭菲菲 田克恭 CHEN Xiao;LI Weiguo;SONG Huanhuan;SU Xiaorui;WANG Tongyan;Tan Feifei;Tian Kegong(National Research Center For Veterinary Medicine,Luoyang 471000,China;Public Bioengineering Co.,Ltd.,Luoyang 471000,China)

机构地区：[1]国家兽用药品工程技术研究中心,洛阳471000 [2]普莱柯生物工程股份有限公司,洛阳471000

出　　处：《中国动物传染病学报》2024年第2期49-57,共9页Chinese Journal of Animal Infectious Diseases

基　　金：郑洛新国家自主创新示范区创新引领型产业集群专项(201200211200);云南省田克恭专家工作站(2019IC062)。

摘　　要：为建立一套表达猪瘟病毒E2蛋白的重组杆状病毒反应器工艺,提高反应器上猪瘟病毒E2蛋白的表达量,以商业化培养基为基础,通过对关键营养组分添加量的DOE试验设计优化,获得一种组合补料配方,并在3 L反应器上对补料方式进行优化,以最大限度提高细胞密度。进一步在反应器上摸索了通气策略,以精确调控猪瘟E2重组杆状病毒培养过程的营养代谢平衡,解决副产物乳酸和氨过度积累导致细胞活力下降的问题。首次使用半连续补料工艺与渗透压动态平衡相结合的补料策略以及定向调节通气的工艺模式,使猪瘟病毒E2蛋白的表达量由130 mg/L增加至520 mg/L,提高了3倍。将3 L反应器工艺在100 L反应器上进行了连续3个批次的验证,结果猪瘟病毒E2蛋白的表达量均大于500 mg/L,收获猪瘟E2蛋白制备的疫苗抗体均在2周后转阳,且抗体水平持续上升,接种后8周抗体阻断率仍维持在80%左右。综上,3 L反应器工艺能够稳定放大至100 L反应器,建立了表达猪瘟病毒E2蛋白的重组杆状病毒100 L反应器工艺,且100 L反应器上重组蛋白的表达量稳定、免疫原性良好。In order to improve the bioreactor process of the recombinant baculovirus expressing classical swine fever virus(CSFV)E2 protein,maximize cell density and increase the expression of CSFV E2 protein,a combined feeding formula was obtained based on the commercial culture medium by optimizing the DOE experimental design of the addition amount of key nutritional components and then the feeding model was optimized on a 3 L bioreactor.The aeration strategy of the bioreactor was further explored to accurately regulate the nutritional and metabolic balances in the culture process of CSFV E2 recombinant baculovirus and solve the problem of the decline of cell viability caused by the excessive accumulation of by-products lactic acid and ammonia.The feeding strategy of semicontinuous feeding process combined with dynamic balance of osmotic pressure and the process mode of directional regulated ventilation were used for the first time to reduce the expression of CSFV E2 protein from 130 mg/L to 520 mg/L,a increase by 3 times.The 3 L bioreactor process was verifi ed in 100 L bioreactor for three consecutive batches.The results showed that the expression of E2 protein was more than 500 mg/L.The vaccine prepared from the harvested CSFV E2 protein induced positive antibody response after 2 weeks of immunization.The antibody level continued to rise and remained at about 80%blocking rate at 8 weeks post inoculation.In summary,the 3 L bioreactor process could be amplifi ed to 100 L bioreactor with the stable expression of CSFV E2 protein and good maintenance of its immunogenicity.

关 键 词：猪瘟病毒 E2蛋白 重组杆状病毒 关键营养单元 半连续补料 定向调节 

分 类 号：S852.65[农业科学—基础兽医学]