噬菌体Qβ病毒样颗粒的制备、纯化及鉴定

doi:10.13523/j.cb.2103034

中国生物工程杂志

2021, Vol. 41

Issue (7): 42-49 DOI: 10.13523/j.cb.2103034

技术与方法

噬菌体Qβ病毒样颗粒的制备、纯化及鉴定

陈修月,周文锋,何庆,苏冰,邹亚文(

)

湖南农业大学动物医学院兽用蛋白质工程疫苗湖南省重点实验室兽用疫苗逆向创制湖南省工程研究中心长沙 410128

Preparation, Purification and Identification of Bacteriophage Qβ Virus-like Particles

CHEN Xiu-yue,ZHOU Wen-feng,HE Qing,SU Bing,ZOU Ya-wen(

)

College of Veterinary Medicine, Hunan Agricultural University, Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, Research & Development Center for Animal Reverse Vaccinology of Hunan Province, Changsha 410128, China

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摘要：

目的: 利用大肠杆菌原核表达系统制备噬菌体Qβ VLPs,验证其自组装能力,纯化后免疫动物以确定其免疫原性,同时制备兔多克隆抗体验证其在哺乳动物细胞中内化能力。方法: 合成pET-28-Qβ-CP质粒,利用大肠杆菌原核表达系统制备Qβ VLPs,通过蔗糖密度梯度离心纯化VLPs,将经凝胶层析柱Sephacryl S-400纯化的Qβ VLPs用透射电镜观察颗粒形态;纯化后的Qβ VLPs加入或不加入佐剂分别免疫新西兰兔,获得血清后用Protein G纯化得到兔多克隆抗体,通过Western blot确定制备的兔多克隆抗体特异性,并利用间接免疫荧光法对Qβ VLPs的细胞内化能力进行鉴定。结果: 制备并获得纯度较高的Qβ VLPs,通过透射电镜观察到大量直径约为28 nm的颗粒;Western blot结果表明制备的兔多克隆抗体能特异性识别Qβ VLPs,且在免疫实验中加入佐剂与不加入佐剂分别免疫动物,对抗体水平的影响不显著。间接免疫荧光法结果表明Qβ VLPs在哺乳动物细胞中具有内化能力。结论: 成功制备Qβ VLPs为后续研发以噬菌体Qβ VLPs为载体的相关疫苗奠定基础。

关键词： 噬菌体Qβ; 病毒样颗粒; 多克隆抗体; 细胞内化

Abstract:

Objective: The self-assembly ability of phage Qβ virus-like particles (VLPs),prepared by Escherichia coli (E. coli) prokaryotic expression system was verified. To determine their immunogenicity, New Zealand rabbits were immunized with purified Qβ VLPs, and them ability to enter mammalian cells was also detected.Methods: The pET-28-Qβ-CP plasmid was constructed, and Qβ VLPs were produced by E. coli expression system. Qβ VLPs were purified by sucrose density gradient centrifugation and gel filtration (Sephacryl S-400 column). The particle morphology of Qβ VLPs were observed by transmission electron microscopy (TEM). Antisera were obtained from the rabbits immunized with the VLPs, along with or without adjuvant, and subsequently, antibodies were purified by Protein G resin. The specificity was determined by Western blot. The indirect immunofluorescence assay (IFA) was used to detect the entry of Qβ VLPs into cells.Results: High purity Qβ VLPs were obtained. TEM results showed that substantial VLPs were observed with diameter of about 28 nm. Western blot showed that Qβ VLPs were specifically recognized by anti-rabbit polyclonal antibodies. Adjuvant had no negative effect on the production of antibody. Based on IFA, the results showed that Qβ VLPs entered various mammalian cells.Conclusion: Qβ VLPs were successfully produced in this study, and they would have great potential as carriers to facilitate the development of future vaccine based on Qβ VLPs.

Key words: Bacteriophage Qβ Virus-like particles Polyclonal antibody Cell entry

收稿日期: 2021-03-15 出版日期: 2021-08-03

ZTFLH:

Q819

通讯作者: 邹亚文 E-mail: yawenzou@stu.hunau.edu.cn

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引用本文:

陈修月,周文锋,何庆,苏冰,邹亚文. 噬菌体Qβ病毒样颗粒的制备、纯化及鉴定[J]. 中国生物工程杂志, 2021, 41(7): 42-49.

CHEN Xiu-yue,ZHOU Wen-feng,HE Qing,SU Bing,ZOU Ya-wen. Preparation, Purification and Identification of Bacteriophage Qβ Virus-like Particles. China Biotechnology, 2021, 41(7): 42-49.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2103034 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I7/42

图1 Qβ外壳蛋白的可溶性分析

图2 Qβ VLPs的纯化结果

图3 Qβ VLPs经Sephacryl S-400分子筛凝胶层析

图4 Qβ VLPs的透射电镜形态

图5 兔多克隆抗体的Western blot分析

图6 间接免疫荧光结果

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