Expression and Purification of the Capsid Protein of Chicken Anemia  Virus and Preparation of Antibody

WANG Xiao-yan; WEN Gang; GAO Yu-long; GAO Hong-lei; WANG Xiao-mei

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October 2006

WANG Xiao-yan, WEN Gang, GAO Yu-long, GAO Hong-lei and WANG Xiao-mei. Expression and Purification of the Capsid Protein of Chicken Anemia Virus and Preparation of Antibody[J]. Virologica Sinica, 2006, 21(5): 477-480.

Citation: WANG Xiao-yan, WEN Gang, GAO Yu-long, GAO Hong-lei, WANG Xiao-mei. Expression and Purification of the Capsid Protein of Chicken Anemia Virus and Preparation of Antibody .VIROLOGICA SINICA, 2006, 21(5) : 477-480.

鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

王晓艳 ¹ ,
文刚 ² ,
高玉龙 ¹ ,
高宏雷 ¹ ,
王笑梅 ^1*,,

1.
1.中国农业科学院哈尔滨兽医研究所，黑龙江哈尔滨 150001
2.
哈尔滨工业大学海洋学院，山东威海 264209

通讯作者： 王笑梅,

摘要

利用PCR技术，以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因（VP1），以T4多聚核苷酸激酶磷酸化处理、纯化后，克隆至表达载体pET-30a(+) 中，从而构建了原核表达质粒pET30-VP1。将pET30-VP1转化至感受态细胞E.coli BL21(DE3)中，经IPTG诱导后，SDS-PAGE分析，可见约45kDa的目的蛋白获得表达。该蛋白经亲和层析纯化后，免疫6-8w的雌性Balb/c鼠，三次免疫后，采血分离血清，制得抗VP1的多克隆血清。以纯化的VP1为包被抗原，用ELISA方法检测，制备的血清效价达12800×以上。以Western blot 检测，该血清可与目的蛋白发生特异性反应，证明其具有良好的免疫原性。VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础。
- 鸡贫血病毒
- , 衣壳蛋白
- , 表达
- , 纯化
- , 多克隆抗体

Expression and Purification of the Capsid Protein of Chicken Anemia Virus and Preparation of Antibody

1.
1.Harbin Veterinary Research Institute, CAAS, Harbin 150001, China
2.
Harbin Institute of Technology, Weihai 264209, China

Corresponding author: WANG Xiao-mei,

Abstract

The coding region of capsid protein gene from Chicken Anemia virus(CAV) was amplified from pPro-VP1 by PCR and cloned into pET-30a（+）. E.coli BL21（DE3）were transformed by the recombinant plasmid pET30-VP1. Analysis by SDS-PAGE and Western blot showed that the target gene was expressed successfully in the form of inclusion body when induced with IPTG. The protein was then purified by Ni2+-affinity chromatography and used to immunize female Balb/c mice. After three rounds of immunizations, antiserum was collected and used to detect the specificity of recombinant protein. ELISA showed that the titer of antiserum was above 1:12800. Moreover, antiserum reacted specifically with purified recombinant protein in Western blot. This study laid a foundation for the development of CAV diagnostic kit and vaccine.
- Chicken anemia virus
- , Capsid protein
- , Expression
- , Purification
- , Polyclonal antibody

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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 and Purification of the Capsid Protein of Chicken Anemia Virus and Preparation of Antibody

Corresponding author: WANG Xiao-mei,

1. 1.Harbin Veterinary Research Institute, CAAS, Harbin 150001, China
2. Harbin Institute of Technology, Weihai 264209, China

Abstract: The coding region of capsid protein gene from Chicken Anemia virus(CAV) was amplified from pPro-VP1 by PCR and cloned into pET-30a（+）. E.coli BL21（DE3）were transformed by the recombinant plasmid pET30-VP1. Analysis by SDS-PAGE and Western blot showed that the target gene was expressed successfully in the form of inclusion body when induced with IPTG. The protein was then purified by Ni2+-affinity chromatography and used to immunize female Balb/c mice. After three rounds of immunizations, antiserum was collected and used to detect the specificity of recombinant protein. ELISA showed that the titer of antiserum was above 1:12800. Moreover, antiserum reacted specifically with purified recombinant protein in Western blot. This study laid a foundation for the development of CAV diagnostic kit and vaccine.