指数补料联合升温发酵法提高治疗性HPV DNA疫苗产量的研究进展

中国医药导刊 ›› 2024, Vol. 26 ›› Issue (8): 748-754.

指数补料联合升温发酵法提高治疗性HPV DNA疫苗产量的研究进展

李晓丽¹，张晓朋¹，刘磊¹，李明珠²，赵超²，张秀军^1*，魏丽惠^2*

1.北京循生免疫医学转化研究院，北京 102600；
2.北京大学人民医院妇产科，北京 100044

收稿日期:2024-11-06 出版日期:2024-08-28 发布日期:2024-08-28
基金资助:
国家重点研发计划（2021YFC2701202）；北京市科技计划（Z231100004823028）

Improvement of the Yield of Therapeutic HPV DNA Vaccine by Exponential Feeding Combined with Temperature Rise Fermentation

1.Aeonvital Institute of Clinical and Translational Immunology， Beijing 102600， China；
2.Department of Obstetrics and Gynecology， Peking University People's Hospital， Beijing 100044， China

Received:2024-11-06 Online:2024-08-28 Published:2024-08-28

摘要/Abstract

摘要：

宫颈癌是全球女性第四大常见癌症。目前上市的HPV疫苗均为预防性疫苗，对已感染HPV人群无治疗效果。HPV感染率仍然很高。近年来，科学家日益关注治疗性HPV疫苗的研发，可诱导细胞免疫杀死已感染HPV的细胞。治疗性HPV DNA疫苗以其低成本、稳定性好的特点备受青睐。为降低治疗性HPV DNA疫苗的成本，目前多采用大肠杆菌高密度发酵生产。本研究使用具有热敏感特性的DNA质粒特性的治疗性HPV DNA疫苗，采用摇瓶培养载有DNA质粒的大肠杆菌，30 ℃培养一定时间后，分别升温至37 ℃或42 ℃，结果显示，升温至42 ℃组比升温至37 ℃组的质粒产量明显升高；采用发酵方式培养大肠杆菌，30 ℃培养一定时间后，升温至42 ℃进一步提高质粒的拷贝数；分别探究恒速补料和指数补料两种方式联合升温发酵策略对DNA质粒产量的影响，结果显示，相较于恒速补料，指数补料可显著提高升温发酵过程中DNA质粒的产量，从125.76 mg·L^-1提升至619.94 mg·L^-1。发酵得到的治疗性HPV DNA疫苗可在293T细胞中高效表达，并能在体内引起HPV抗原特异性细胞免疫反应。本研究认为指数补料联合升温发酵策略对其他DNA质粒的发酵也有一定的借鉴意义，也为CAR-T等细胞产品、RNA相关产品提供高质量DNA质粒提供参考。

关键词: 人乳头瘤病毒（HPV）, 治疗性HPV DNA疫苗, 热敏型质粒, 升温发酵, 恒速补料, 指数补料

Abstract:

Cervical cancer is the fourth most common cancer in women worldwide. HPV vaccines currently on the market are all prophylactic vaccines， which have no therapeutic effect on people already infected with HPV. HPV infection rates remain high. Scientists are developing therapeutic HPV vaccines that induce cellular immunity to kill cells infected with HPV in rencent years. Therapeutic HPV DNA vaccines are favored for its low cost and good stability. In order to reduce the cost of therapeutic HPV DNA vaccines， high density fermentation of E. coli is used. This study used a therapeutic HPV DNA vaccine， which is a DNA plasmid with heat-sensitive property. Firstly， E. coli with DNA plasmid was cultured in shake. After culture at 30 ℃ for a certain time， the temperature was raised to 37 ℃ or 42 ℃. It was found that the plasmid yield of the group with temperature rising to 42 ℃ was significantly higher than that of the group with temperature rising to 37 ℃. Further research was conducted to culture E. coli by fermentation. After culture at 30 ℃ for a certain time， the temperature was raised to 42 ℃ to further improve the copy number of the plasmid， and to explore the influence of temperature rise fermentation strategy combined with constant feeding or exponential feeding on the yield of DNA plasmid.The results showed that compared with constant feeding， exponential feeding could significantly increase the yield of DNA plasmid combining with the temperature rise fermentation strategy， from 125.76 mg·L^-1 to 619.94 mg·L^-1. The therapeutic HPV DNA vaccine obtained by fermentation is highly expressed in 293T cells and can induce a strong HPV specific cellular immune response in vivo. The exponential feeding combined with temperature rise fermentation strategy also has reference significance for the fermentation of other DNA plasmids， and can provide reference on high-quality DNA plasmids for cell products such as CAR-T and RNA related products.

Key words: HPV； , Therapeutic HPV DNA vaccine； , Heat-sensitive plasmid； , Temperature rise fermentation strategy； , Constant feeding； , Exponential feeding

中图分类号:

R737.33

李晓丽, 张晓朋, 刘磊, 李明珠, 赵超, 张秀军, 魏丽惠.

指数补料联合升温发酵法提高治疗性HPV DNA疫苗产量的研究进展 [J]. 中国医药导刊, 2024, 26(8): 748-754.

LI Xiaoli, ZHANG Xiaopeng, LIU Lei, LI Mingzhu, ZHAO Chao, ZHANG Xiujun, WEI Lihui.

Improvement of the Yield of Therapeutic HPV DNA Vaccine by Exponential Feeding Combined with Temperature Rise Fermentation [J]. CHINESE JOURNAL OF MEDICINAL GUIDE, 2024, 26(8): 748-754.

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