Eukaryotic expression of human metapneumovirus fusion protein in insect cells by using a baculovirus system

doi:10.13406/j.cnki.cyxb.2007.05.004

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Eukaryotic expression of human metapneumovirus fusion protein in insect cells by using a baculovirus system

更新时间：2024-01-17

- Eukaryotic expression of human metapneumovirus fusion protein in insect cells by using a baculovirus system
- Journal of Chongqing Medical University Issue 5, Pages: 455-458(2007)
- 作者机构：
  
  重庆医科大学附属儿童医院P2实验室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2007.05.004
  CLC： R346
- Published：2007，
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[1]张琴,赵晓东,毛华伟,杨锡强.人类偏肺病毒F基因真核表达质粒的构建及表达[J].重庆医科大学学报,2007(05):455-458+502.

Eukaryotic expression of human metapneumovirus fusion protein in insect cells by using a baculovirus system. [J]. Journal of Chongqing Medical University (5):455-458(2007)
[1]张琴,赵晓东,毛华伟,杨锡强.人类偏肺病毒F基因真核表达质粒的构建及表达[J].重庆医科大学学报,2007(05):455-458+502. DOI： 10.13406/j.cnki.cyxb.2007.05.004.

Eukaryotic expression of human metapneumovirus fusion protein in insect cells by using a baculovirus system. [J]. Journal of Chongqing Medical University (5):455-458(2007) DOI： 10.13406/j.cnki.cyxb.2007.05.004.

摘要

目的:构建人类偏肺病毒融合蛋白（F）基因的真核表达质粒

并在昆虫细胞sf-9中表达。方法:采用RT-PCR法扩增人类偏肺病毒A亚型CAN97-83 F基因的全长序列

将其插入供体质粒pFastBacHT载体多克隆位点区

经酶切鉴定后转化大肠杆菌DH10bac。供体质粒与受体杆粒（Bacmid）发生位点特异性转座获得重组的杆粒。采用脂质体将重组杆粒转染进昆虫细胞sf-9

包装获得感染性杆状病毒。用此病毒感染sf-9细胞并表达蛋白

经6×His树脂纯化后用免疫印迹法验证表达蛋白。结果:PCR及序列分析证实所获得的重组杆粒包含hMPV全长F基因序列

被重组杆状病毒感染的昆虫细胞sf-9显示典型细胞病变。采用抗6×His单克隆抗体为一抗

免疫印迹法检测到分子量约70ku的较单一的蛋白条带

证实F蛋白表达成功。结论:杆状病毒是一高通量、目标蛋白抗原性保存完好的表达系统。采用这一系统成功表达的hMPV F蛋白

将为我国hMPV血清流行病学和免疫发病机理研究奠定基础。

Abstract

Objective:To construct the eukaryotic recombinant bacmid containing fusion protein gene of human metapneumovirus and express it in insect cells.Methods:The full-length fusion protein gene of human metapneumovirus was amplified by re- verse transcriptional polymerase chain reaction(RT-PCR).After inserted at the multiple clone site of donor plasmid

pFast- BacHT

target gene was delivered to bacmid to yield a recombinant bacmid.This recombinant baemid was subsequently pack- aged in sf-9 cells and infectious recombinant baculovirus was used to inoculate fresh sf-9 cells to express fusion protein.Ex- pressed protein was purified by using a 6xHis tag rasin and identified by western bloning.Result:Nucleotide sequence analy- sis of PCR products amplified from rBacmid-hMPVF showed 100% identity to public sequence of CAN 97-83 F gene se- quence.Sf-9 cells infected by recombinant baculovirus displayed typical cytopathic effects.By using 6xHis tag monoclonal an- tibody and western blotting

a single band with molecular weight around 70ku was identified.Conclusion:The fusion protein of hMPV is successfully expressed by using baculovirus expression system in insect cells

which will benefit the study on sero-epidemiology and pathogenesis of hMPV infection in the future.

关键词

人类偏肺病毒融合蛋白真核表达杆状病毒

Keywords

Human metapneumovirufusion proteinEukaryotic expressionbaculovirus

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