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无野田村病毒感染的H5昆虫细胞株筛选及鉴定
郭兆隆,杨施瑜,刘喜凤
0
(北京鼎持生物技术有限公司)
摘要:
昆虫细胞High five(H5)常用于重组杆状病毒表达外源蛋白,表达的外源蛋白可应用于疫苗和抗体等生物制品的开发,但是目前使用的商业化H5昆虫细胞经检测含有外源病毒野田村病毒(Nodavirus,NV),可能会影响生物制品的安全性。本研究通过对H5单克隆细胞株的培养,经PCR鉴定NV的特异性条带,筛选出NV阴性细胞克隆株,经多轮验证,最终确定5株NV阴性的H5-WN细胞株。这5株细胞株在传代40代后PCR检测,NV依旧为阴性。温度和杆状病毒不会诱导NV再次感染。5株无NV污染细胞株与原始细胞株H5相比增殖特性无明显差别。对猪圆环CAP蛋白重组杆状病毒在5株H5-WN细胞和H5细胞中蛋白表达比较,表明筛选出的5株H5-WN细胞株CAP表达量与H5相似。由于该细胞株无NV感染,剔除NV后细胞株的增殖特性和蛋白表达不受影响,从安全性和纯净性的角度考虑,无NV感染的H5-WN细胞株更适合重组杆状病毒外源蛋白的表达。
关键词:  昆虫细胞  H5  NV  杆状病毒  增殖特性
DOI:
投稿时间:2020-05-24修订日期:2020-07-15
基金项目:
Screening and Identification of H5 Insect Cell Lines without Nodavirus Infection
(Beijing Dingzhi Biotechnology Co.,Ltd.,Beijing,10081)
Abstract:
The insect cell line High Five (H5) is frequently used to express recombinant proteins using the baculovirus expression system. Those proteins could be used in vaccine and antibody preparation. However, H5 cells could be latently infected with a nodavirus (NV) which may affect the safety of biological products. In this study, 5 H5-WN cell lines without NV infection were screened and determined by PCR. After 40 passages, 5 cell screening H5-NV lines were still free of NV. Temperature and Baculovirus couldn’t induce the reinfection of NV. There was no significant difference between 5 NV free cell lines and the original cell lines H5 in the proliferation capacity. At the same time, this study also compared the porcine circovirus CAP protein expression in 5 cell lines and H5. The results showed that the expression of CAP protein in 5 cell lines free of NV was similar with that in H5. Since these cell lines have no NV infection, they are more suitable for the expression of foreign proteins from the perspective of safety and purity.
Key words:  insect cells  H5  NV  baculovirus  proliferation capacity

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