猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定

doi:10.16431/j.cnki.1671-7236.2016.07.033

《中国畜牧兽医》---唯一指定的官方网站 ›› 2016, Vol. 43 ›› Issue (7): 1876-1883.doi: 10.16431/j.cnki.1671-7236.2016.07.033

猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定

郭慧娟¹, 樊翠¹, 张英¹, 吕茂杰¹, 梁武^1,2, 杨保收¹

1. 天津瑞普生物技术股份有限公司, 瑞普生物研究院, 天津 300308;
2. 中国农业大学动物医学院, 北京 100193

收稿日期:2015-12-24 出版日期:2016-07-20 发布日期:2016-07-22
通讯作者: 杨保收 E-mail:bsyang@ringpu.com
作者简介:郭慧娟(1987-),女,山西太原人,硕士,主要从事生物制品研发工作,E-mail:ghjanna@126.com
基金资助:
瑞普生物研究院动物疫病流行病学调查专项基金（RPBVI20130010）

Expression and Identification of Porcine Circovirus type 2 Capsid Protein Using flashBAC Baculovirus Expression System

GUO Hui-juan¹, FAN Cui¹, ZHANG Ying¹, LV Mao-jie¹, LIANG Wu^1,2, YANG Bao-shou¹

1. Ringpu Biological Institute, Tianjin Ringpu Biological Technology Co., Ltd., Tianjin 300308, China;
2. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

Received:2015-12-24 Online:2016-07-20 Published:2016-07-22

摘要/Abstract

摘要：

试验旨在通过真核表达系统表达猪圆环病毒2型（porcine circovirus type 2，PCV2） CAP蛋白。以PCV2 TZ0601株为模板，将PCV2 CAP全基因及CAP去除信号肽的基因编码序列克隆至pOET3载体上，酶切与测序鉴定正确后，将重组质粒pOET3-CAP及pOET3-CAP-X转染sf9昆虫细胞。采用flashBAC杆状病毒表达系统表达PCV2 CAP及去除信号肽的CAP蛋白，通过间接免疫荧光法、SDS-PAGE 和Western blotting鉴定目的蛋白的表达。结果表明，真核表达质粒pOET3-CAP及pOET3-CAP-X构建成功，目的基因在sf9昆虫细胞中高效表达，得到的蛋白经SDS-PAGE和Western blotting鉴定，在25~35 ku处有蛋白条带，表达的蛋白质可被PCV2阳性血清识别。试验结果为进一步制备PCV2亚单位疫苗及诊断抗原试剂盒的研发奠定了基础。

关键词: 猪圆环病毒2型; flashBAC杆状病毒表达系统; CAP蛋白

Abstract:

The study was aimed to express CAP protein of porcine circovirus type 2 (PCV2) by eukaryotic expression system.The PCV2 TZ0601 strain was the template,the CAP protein with or without the signal peptide of PCV2 coding sequence were cloned into pOET3 vector.The constructed plasmid were confirmed by restriction enzyme digestion and DNA sequencing,then sf9 insect cells were transfected with recombinant plasmid pOET3-CAP and pOET3-CAP-X.The test was designed to express the CAP protein with or without the signal peptide of PCV2 by flashBAC baculovirus expression system.Expression of PCV2 gene were confirmed by indirect immunofluorescent assay (IFA),SDS-PAGE and Western blotting.The results showed that the eukaryotic expression plasmids of pOET3-CAP and pOET3-CAP-X were constructed successfully and the gene was highly expressed in sf9 insect cells.After expression,we could see our target band about 25 to 35 ku with SDS-PAGE and Western blotting.The immuno-reactivity of the protein was confirmed by antisera against PCV2.This would lay a foundation for a further study of PCV2 subunit vaccine and diagnostic antigen kit.

Key words: porcine circovirus type 2; flashBAC baculovirus expression system; CAP protein

中图分类号:

S852.65⁺9.2

郭慧娟, 樊翠, 张英, 吕茂杰, 梁武, 杨保收. 猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2016, 43(7): 1876-1883.

GUO Hui-juan, FAN Cui, ZHANG Ying, LV Mao-jie, LIANG Wu, YANG Bao-shou. Expression and Identification of Porcine Circovirus type 2 Capsid Protein Using flashBAC Baculovirus Expression System[J]. , 2016, 43(7): 1876-1883.

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猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定

Expression and Identification of Porcine Circovirus type 2 Capsid Protein Using flashBAC Baculovirus Expression System

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