Cloning and Expression of Phospholipase A2 Domain of Periplaneta fuliginosa Densovirus in E.coli

YU Hai-yang; ZHANG Jia-min; YANG Bo; JIANG Hong; ZHOU Liang; LU Jie; CHENG Wu-guo; LI Dou-lin; HU Yuan-yang*

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April 2006

YU Hai-yang, ZHANG Jia-min, YANG Bo, JIANG Hong, ZHOU Liang, LU Jie, CHENG Wu-guo, LI Dou-lin and HU Yuan-yang*. Cloning and Expression of Phospholipase A2 Domain of Periplaneta fuliginosa Densovirus in E.coli[J]. Virologica Sinica, 2006, 21(2): 181-185.

Citation: YU Hai-yang, ZHANG Jia-min, YANG Bo, JIANG Hong, ZHOU Liang, LU Jie, CHENG Wu-guo, LI Dou-lin, HU Yuan-yang*. Cloning and Expression of Phospholipase A2 Domain of Periplaneta fuliginosa Densovirus in E.coli .VIROLOGICA SINICA, 2006, 21(2) : 181-185.

黑胸大蠊浓核病毒磷脂酶A2功能区的克隆及其表达

1.
武汉大学生命科学学院病毒学国家重点实验室，湖北武汉 430072

摘要

通过RT-PCR 扩增获得PfDNV 结构蛋白基因VP1 含磷脂酶A2（PL A2）功能区片段，将其连接到pMD18-T 载体上并亚克隆到原核表达载体pET28a 和pET26b，构建阅读框架正确的重组表达载体pET28a-PLA 和 pET26b-PLA，转化大肠杆菌BL21-codonplus（DE3）-RIL，经IPTG 诱导， SDS-PAGE 显示得到了目的融合蛋白，以抗组氨酸的单克隆抗体对经Ni-NTA 亲和层析柱纯化的目的蛋白进行了western blot 鉴定，结果表明成功表达 PfDNV 结构蛋白PLA2，对于研究该酶的生物学特性及其在病毒对细胞侵染过程中的功能奠定了基础。
- 黑胸大蠊浓核病毒（PfDNV）
- , 磷脂酶A2
- , 原核表达

Cloning and Expression of Phospholipase A2 Domain of Periplaneta fuliginosa Densovirus in E.coli

1.
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China

Abstract

The phospholipase A2 functional domain of PfDNV capsid gene VP1 was obtained by RT-PCR amplification. The amplified fragment was ligated into a pMD18-T vector and sub-cloned into prokaryotic expression vector pET28a and pET26b. The recombinant plasmid pET28a-PLA and pET26b-PLA were used to transform E. coli BLl21-codonplus（DE3）-RIL competent cells. After induction by IPTG, SDS-PAGE indicated that the highly expressed fusion protein was produced. The fusion protein was purified with Ni-NTA affinity columns and analyzed by Western blot using mouse anti-His monoclonal antibodies. The results demonstrated that the recombinant PLA2 protein of PfDNV capsid gene was successfully expressed, which should facilitate further studies on the biological properties of the enzyme and its function in virus infection.
- Periplaneta fuliginosa densovirus（PfDNV）
- , phospholipase A2
- , Prokaryotic expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning and Expression of Phospholipase A2 Domain of Periplaneta fuliginosa Densovirus in E.coli

1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China

Abstract: The phospholipase A2 functional domain of PfDNV capsid gene VP1 was obtained by RT-PCR amplification. The amplified fragment was ligated into a pMD18-T vector and sub-cloned into prokaryotic expression vector pET28a and pET26b. The recombinant plasmid pET28a-PLA and pET26b-PLA were used to transform E. coli BLl21-codonplus（DE3）-RIL competent cells. After induction by IPTG, SDS-PAGE indicated that the highly expressed fusion protein was produced. The fusion protein was purified with Ni-NTA affinity columns and analyzed by Western blot using mouse anti-His monoclonal antibodies. The results demonstrated that the recombinant PLA2 protein of PfDNV capsid gene was successfully expressed, which should facilitate further studies on the biological properties of the enzyme and its function in virus infection.