Expression of HCV Structural Proteins and Self-assembly of HCV-like Particles in Insect Cells

WANG Rui-qing; LIANG Chang-yong; JIAO Cheng-song; SONG Jian-hua; CHEN Xin-wen

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February 2005

WANG Rui-qing, LIANG Chang-yong, JIAO Cheng-song, SONG Jian-hua and CHEN Xin-wen. Expression of HCV Structural Proteins and Self-assembly of HCV-like Particles in Insect Cells[J]. Virologica Sinica, 2005, 20(1): 5-10.

Citation: WANG Rui-qing, LIANG Chang-yong, JIAO Cheng-song, SONG Jian-hua, CHEN Xin-wen. Expression of HCV Structural Proteins and Self-assembly of HCV-like Particles in Insect Cells .VIROLOGICA SINICA, 2005, 20(1) : 5-10.

HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的组装

王瑞青 ^1,2 ,
梁昌镛 ^1,2 ,
焦成松 ³ ,
宋建华 ^1,2 ,
陈新文 ^1*

1.
1.中国科学院武汉病毒研究所分子病毒学重点实验室湖北武汉430071 2.中国科学院研究生院北京湖北武汉 3.中国人民解放军广州军区武汉总医院

摘要

根据GenBank公布的日本脑炎病毒(Japanese Encephalitis Virus, JEV) SA14 14 2株的核酸序列和人流感病毒的血凝素基因(ha)序列, 设计一对特异性引物, 用PCR方法扩增编码JEV囊膜蛋白主要抗原域基因, 其中含ha基因主要核苷酸序列。将PCR产物定向克隆入原核表达载体pET 32a(+), 构建原核表达载体pET EHA。阳性质粒转化BL21(DE3)宿主菌, 经IPTG诱导获得表达, 重组蛋白以包涵体的形式存在。Western blot分析表明表达产物具有良好的免疫学活性。利用纯化的表达产物与流感病毒血凝素单抗及乳胶建立了诊断日本脑炎病毒抗体水平的乳胶凝集试验。结果表明乳胶凝集方法具有简便快速、敏感性高、特异性强、价格低廉、可现场检测等优点, 是一种适合基层兽医单位用于日本脑炎病毒抗体水平检测的新方法。
- 丙型肝炎病毒
- , 结构蛋白
- , 表达
- , 病毒样颗粒
- , Sf21细胞

Expression of HCV Structural Proteins and Self-assembly of HCV-like Particles in Insect Cells

Abstract

Using a pair of specific primers designed according to the nucleotide sequence of Japanese encephalitis virus and Hameoaggulatinin of Human influenza virus from GenBank, the gene of JEV E protein’s main antigenic domain including the sequence of Human influenza virus ha gene was amplified with PCR method. The PCR product was successively cloned into pET-32a(+) vector to get a prokaryotic expression plasmid pET-EHA. After the positive plasmid was transformed into the host cell BL21(DE3) , the target gene was successfully expressed in the form of inclusion bodies when induced with IPTG. Western-blotting analysis proved the recombinant protein has good reactivity ability against JEV antibodies. The Latex Agglutination Test (LAT) method for the detection of JEV antibodies was established with the purified recombinant protein. The result indicated that LAT was a simple, rapid, sensitive, specific, inexpensive method which is suitable for detecting antibodies against JEV in serum and serological survey of JE .
- Hepatitis C virus （HCV）
- , Structural proteins
- , Expression
- , Virus-Like Particles
- , Sf21 cell

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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

Expression of HCV Structural Proteins and Self-assembly of HCV-like Particles in Insect Cells

Abstract: Using a pair of specific primers designed according to the nucleotide sequence of Japanese encephalitis virus and Hameoaggulatinin of Human influenza virus from GenBank, the gene of JEV E protein’s main antigenic domain including the sequence of Human influenza virus ha gene was amplified with PCR method. The PCR product was successively cloned into pET-32a(+) vector to get a prokaryotic expression plasmid pET-EHA. After the positive plasmid was transformed into the host cell BL21(DE3) , the target gene was successfully expressed in the form of inclusion bodies when induced with IPTG. Western-blotting analysis proved the recombinant protein has good reactivity ability against JEV antibodies. The Latex Agglutination Test (LAT) method for the detection of JEV antibodies was established with the purified recombinant protein. The result indicated that LAT was a simple, rapid, sensitive, specific, inexpensive method which is suitable for detecting antibodies against JEV in serum and serological survey of JE .