Retroviral Vector Mediated HCV E1E2 Envelope Protein Expression in Eukaryotic Cells and Animal Immune Experiment

XU Xin-gang; HU Jian-he; ZHANG Yan-ming; DENG Hong-kui

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December 2004

XU Xin-gang, HU Jian-he, ZHANG Yan-ming and DENG Hong-kui. Retroviral Vector Mediated HCV E1E2 Envelope Protein Expression in Eukaryotic Cells and Animal Immune Experiment[J]. Virologica Sinica, 2004, 19(6): 563-567.

Citation: XU Xin-gang, HU Jian-he, ZHANG Yan-ming, DENG Hong-kui. Retroviral Vector Mediated HCV E1E2 Envelope Protein Expression in Eukaryotic Cells and Animal Immune Experiment .VIROLOGICA SINICA, 2004, 19(6) : 563-567.

逆转录病毒载体介导的HCV囊膜蛋白基因的表达及动物免疫试验

许信刚 ^1,2 ,
胡建和 ² ,
张彦明 ¹ ,
邓宏魁 ^2,,

1.
1.西北农林科技大学动物科技学院，陕西杨凌 712100
2.
北京大学生命科学学院，北京100871

通讯作者： 邓宏魁,

摘要

利用DNA重组技术将丙型肝炎病毒（HCV）H77 株E1E2囊膜蛋白基因插入逆转录病毒载体pBABE-puro 中构建成重组逆转录病毒载体pBABE-puro-E1E2，该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293T细胞中包装逆转录病毒假病毒。用包装的假病毒感染SP2/0细胞，经嘌呤霉素筛选阳性细胞后进行流式细胞技术（FACS）分析，结果表明HCV e1e2基因在SP2/0细胞膜上成功表达。将表达E1E2蛋白的SP2/0细胞腹腔免疫BA LB/c小鼠，经FACS分析免疫鼠血清，成功诱导小鼠产生了抗HCV E1E2蛋白的抗体，Western blot表明该抗体能与原核系统表达的E2蛋白结合。
- 丙型肝炎病毒
- , 囊膜蛋白
- , 逆转录病毒载体
- , 假病毒
- , FACS

Retroviral Vector Mediated HCV E1E2 Envelope Protein Expression in Eukaryotic Cells and Animal Immune Experiment

1.
1.College of Animal Sciences and Technology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China
2.
College of Life Sciences，Peking University，Beijing 100871, China

Corresponding author: DENG Hong-kui,

Abstract

The recombinant retroviral vector pBABE-puro-E1E2 was constructed by inserting the full-length HCV e1e2 gene of H77 strain into pBABE-puro. Both the recombinant retroviral vector and the pVSVg plasmid were transfected into eukaryotic cells 293T by calcium phosphate transfection method. And then, the pseudovirus were produced. The pseudovirus infected eukaryotic cells SP2/0 and E1E2 protein was expressed. E1E2 protein was detected by puromycin-resistant and FACS analysis. BALB/c mice were injected in abdomen with expressing E1E2 protein SP2/0 cells. Anti-HCV E1E2 antibody was screened by FACS. Moreover, the antibody was also analyzed by Western blot using E2 protein antigen which was expressed in E. coli. The results showed that HCV E1E2 protein was expressed in SP2/0 cells’ envelope successfully. FACS could detect specific anti-E1E2 antibody in SP2/0 cells immune mouse serum. Western blot analysis showed that SP2/0 cells immune mouse serum could react specially to E2 protein that was expressed in E.coli.
- Hepatitis C virus
- , Envelope protein gene
- , Retroviral vector
- , Pseudovirus
- , FACS

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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

Retroviral Vector Mediated HCV E1E2 Envelope Protein Expression in Eukaryotic Cells and Animal Immune Experiment

Corresponding author: DENG Hong-kui,

1. 1.College of Animal Sciences and Technology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China
2. College of Life Sciences，Peking University，Beijing 100871, China

Abstract: The recombinant retroviral vector pBABE-puro-E1E2 was constructed by inserting the full-length HCV e1e2 gene of H77 strain into pBABE-puro. Both the recombinant retroviral vector and the pVSVg plasmid were transfected into eukaryotic cells 293T by calcium phosphate transfection method. And then, the pseudovirus were produced. The pseudovirus infected eukaryotic cells SP2/0 and E1E2 protein was expressed. E1E2 protein was detected by puromycin-resistant and FACS analysis. BALB/c mice were injected in abdomen with expressing E1E2 protein SP2/0 cells. Anti-HCV E1E2 antibody was screened by FACS. Moreover, the antibody was also analyzed by Western blot using E2 protein antigen which was expressed in E. coli. The results showed that HCV E1E2 protein was expressed in SP2/0 cells’ envelope successfully. FACS could detect specific anti-E1E2 antibody in SP2/0 cells immune mouse serum. Western blot analysis showed that SP2/0 cells immune mouse serum could react specially to E2 protein that was expressed in E.coli.