5型和35型腺病毒纤毛蛋白的原核表达及活性验证

中国生物工程杂志

2013, Vol. 33

Issue (12): 15-20

研究报告

5型和35型腺病毒纤毛蛋白的原核表达及活性验证

张文峰^1,2, 张琼宇³, 薄华本^1,2, 邵红伟^1,2, 李晓程^1,2, 王腾^1,2, 黄树林^1,2

1. 广东药学院生命科学与生物制药学院广州 510006;
2. 广东省生物技术候选药物研究重点实验室广州 510006;
3. 永州职业技术学院基础医学部永州 425100

Prokaryotic Expression and Activity Detection of Ad5/Ad35 Fiber Gene

ZHANG Wen-feng^1,2, ZHANG Qiong-yu³, BO Hua-ben^1,2, SHAO Hong-wei^1,2, LI Xiao-cheng^1,2, WANG Teng^1,2, HUANG Shu-lin^1,2

1. School of Life Science & Biopharmacology, Guang Dong Pharmaceutical University, Guanzhou 510006, China;
2. Guang Dong Provincial Key Laboratory of Biotechnology Candidate Drug Research, Guangzhou 510006, China;
3. Department of Basic Medical Science, Yongzhou Vocational Technical College, Yongzhou 425100, China

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摘要： 目的：原核表达5型腺病毒纤毛蛋白（Ad5 fiber）和35型腺病毒纤毛蛋白（Ad35 fiber），并对蛋白活性进行初步分析。方法：将重组质粒pET28a-Ad5 fiber和pET28a-Ad35 fiber转化入E.coli BL21（DE3），IPTG诱导表达后，经Ni-NTA凝胶珠亲和纯化，用SDS-PAGE鉴定纯化蛋白，并用蛋白竞争性实验验证纯化蛋白的活性。结果：转化溶原菌后能够表达目的蛋白，Ad35 fiber蛋白对Ad5（5型腺病毒）感染无影响，只能阻断Ad5F35（5型腺病毒纤毛被替换为35型腺病毒纤毛的嵌合腺病毒）对细胞的感染；Ad5 fiber蛋白对嵌合腺病毒Ad5F35感染无影响，只能抑制Ad5对细胞的感染。结论：成功表达具有生物活性的5型和35型腺病毒纤毛蛋白，为进一步研究嵌合腺病毒Ad5F35感染细胞的机制奠定基础。

关键词： 嵌合腺病毒载体; 腺病毒纤毛蛋白; 原核表达

Abstract: Aim: To construct the prokaryotic expression vector for Ad5 fiber and Ad35 fiber genes, purify proteins and study the activity. Methods: Recombinant plasmid pET28a-Ad5 fiber and pET28a-Ad35 fiber were transformed into E.coli BL21(DE3) and the proteins expression were induced with IPTG. The expressed proteins Ad5 fiber and Ad35 fiber were purified by Ni²⁺ affinity chromatography and identified by SDS-PAGE. The bioactivity of proteins Ad5 fiber and Ad35 fiber were certificated by competitive binding method. Results: The proteins Ad5 fiber and Ad35 fiber can be detected in the supernatant. The proteins Ad5 fiber specifically inhibited the infectivity of the Ad5 virus in a dose-dependent manner, while had no effect on the Ad5F35-mediated gene transfer. The proteins Ad35 fiber blocked the Ad5F35-mediated gene transfer in a dose-dependent manner and had no effect on the infectivity of the Ad5 virus in competition experiments. Conclusion: Recombinant vectors Ad5 fiber and Ad35 fiber were constructed successfully. The proteins Ad5 fiber and Ad35 fiber could specifically bind with different receptors. The study will lay a foundation for the investigation on the mechanism of Ad5F35 infection.

Key words: Adenoviral vector Adenovirus fiber protein Prokaryotic expression

收稿日期: 2013-09-06 出版日期: 2013-12-25

ZTFLH:

Q786

基金资助: “重大新药创制” 科技重大专项 “十一五” 计划项目（2009ZX09103-708）；国家自然科学基金（31100664，31300737，81303292）；东莞市科技计划项目（2011105102027）资助项目

通讯作者: 黄树林，E-mail：shulhuang@sina.com E-mail: shulhuang@sina.com

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引用本文:

张文峰, 张琼宇, 薄华本, 邵红伟, 李晓程, 王腾, 黄树林. 5型和35型腺病毒纤毛蛋白的原核表达及活性验证[J]. 中国生物工程杂志, 2013, 33(12): 15-20.

ZHANG Wen-feng, ZHANG Qiong-yu, BO Hua-ben, SHAO Hong-wei, LI Xiao-cheng, WANG Teng, HUANG Shu-lin. Prokaryotic Expression and Activity Detection of Ad5/Ad35 Fiber Gene. China Biotechnology, 2013, 33(12): 15-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I12/15

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