增殖细胞核抗原蛋白在<i>Spodoptera frugiperda</i>昆虫细胞中的表达及纯化 <sup>*</sup>

doi:10.13523/j.cb.20180703

中国生物工程杂志

2018, Vol. 38

Issue (7): 14-20 DOI: 10.13523/j.cb.20180703

研究报告

增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化 ^*

陈军军¹,娄颖¹,张元兴^1,^2,³,刘琴^1,^2,³,刘晓红^1,^2,^**(

)

1 华东理工大学上海 200237
2 上海海洋动物疫苗工程技术研究中心上海 200237
3上海生物制造技术协同创新中心上海 200237

Expression and Purification of Proliferating Cell Nuclear Antigen in Spodoptera frugiperda Cells

Jun-jun CHEN¹,Ying LOU¹,Yuan-xing ZHANG^1,^2,³,Qin LIU^1,^2,³,Xiao-hong LIU^1,^2,^**(

)

1 East China University of Science and Technology, Shanghai 200237, China
2 Shanghai Engineering Research Centers of Maricultured Animal Vaccines, Shanghai 200237, China
3 Shanghai Collaborative Innovation Center for Biomanufacturing, Shanghai 200237, China

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摘要：

目的:利用昆虫细胞表达系统生产重组的人增殖细胞核抗原(proliferating cell nuclear antigen, PCNA),并进行纯化和抗体结合特性鉴定。方法:以HeLa细胞逆转录的cDNA为模板,扩增人PCNA基因,并插入杆状病毒载体AcMNPV。利用昆虫细胞得到PCNA基因的重组杆状病毒。病毒感染细胞表达蛋白,联合镍柱亲和层析和离子交换层析获得高纯度的重组人PCNA蛋白。ELISA法测定抗体结合特异性。结果:以HeLa细胞cDNA为模板得到的基因序列同GenBank的人PCNA基因序列一致。草地贪夜蛾细胞(Spodoptera frugiperda, Sf9)表达重组人PCNA(recombinant human PCNA, rPCNA)的最佳感染值(MOI)和感染时间分别为0.05h和144h。rPCNA的产量高达110mg/L细胞,纯度> 95%。间接ELISA法检测抗体结合特性,rPCNA的敏感性和特异性分别为93.3%和85.0%。结论:建立了rPCNA的表达和纯化方法,获得了高效表达、高度抗体结合特异性的PCNA蛋白,该蛋白质能进一步开发为PCNA相关疾病的体外诊断试剂盒,具较大的应用价值。

关键词： 增殖细胞核抗原; 杆状病毒; 昆虫细胞; 蛋白质纯化; 抗体结合特异性

Abstract:

Objetive: The object is to produce recombinant human proliferating cell nuclear antigen (PCNA) protein using insect cell expression system and purify and identify its antibody binding characterization. Methods: Human PCNA gene was amplified from HeLa cells and cloned into the baculovirus vector AcMNPV. Using insect cells, the recombinant baculovirus containing the PCNA gene was obtained. The virus infected the cells to express the protein, which was reached high purity by combining the nickel column affinity chromatography and the ion exchange chromatography. ELISA method was set to identify its binding activity. Results: Full length recombinant human PCNA (rPCNA) was produced in a baculovirus expression system. The optimal multiplicity of infection (MOI) value and infected time were 0.05h and 144h respectively. The produced protein samples were subsequently purified by a two-step procedure, including Ni-NTA affinity chromatography and ion exchange chromatography. The yield of rPCNA was up to 110mg/L cell culture, with a purity > 95% by SDS-PAGE. Indirect ELISA results showed that antibody binding activity of rPCNA was much higher than that of PCNA expressed by E. coli and rPCNA took a sensitivity and specificity of 93.3% and 85.0%, respectively. Conclusions: An expression and purification procedure for rPCNA and the produced rPCNA presented high antibody binding characterization were established which would have great potential applications on diagnosis of PCNA-associated diseases in vitro.

Key words: Proliferating cell nuclear antigen Baculovirus Insect cells Protein purification Antibody binding characterization

收稿日期: 2018-02-26 出版日期: 2018-08-13

ZTFLH:

Q819

基金资助: 国家自然科学基金(31622059);中央高校基础研究基金(222201717019)

通讯作者: 刘晓红 E-mail: liuxiaohong@ecust.edu.cn

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引用本文:

陈军军,娄颖,张元兴,刘琴,刘晓红. 增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化 ^*[J]. 中国生物工程杂志, 2018, 38(7): 14-20.

Jun-jun CHEN,Ying LOU,Yuan-xing ZHANG,Qin LIU,Xiao-hong LIU. Expression and Purification of Proliferating Cell Nuclear Antigen in Spodoptera frugiperda Cells. China Biotechnology, 2018, 38(7): 14-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180703 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/14

图1 Bac-to-Bac系统生产PCNA的重组杆状病毒

图2 MOI和浸染时间对rPCNA蛋白表达量的影响

图3 小规模表达及纯化rPCNA蛋白

图4 大规模表达和纯化rPCNA蛋白

表1 ELISA检测rPCNA的抗体结合特性

图5 间接ELISA检测rPCNA的抗体结合特异性

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