引用本文:
【打印本页】   【下载PDF全文】   View/Add Comment  Download reader   Close
←前一篇|后一篇→ 过刊浏览    高级检索
本文已被:浏览 2762次   下载 2309 本文二维码信息
码上扫一扫!
分享到: 微信 更多
草鱼出血病病毒VP7蛋白基因的人工合成与原核表达
韦先超1,2, 杨泽晓1,3, 王 印1, 裴腊梅1, 姚学萍1, 汪开毓1, 杨水仙1
1.四川农业大学动物医学院;2.四川省水产学校;3.动物疫病与人类健康四川省重点实验室
摘要:
根据GenBank已发表的GCRV VP7蛋白基因序列, 设计合成2条特异性引物和19条搭桥引物, 通过搭桥PCR人工合成带有3处变异碱基的GCRV VP7蛋白基因整个编码框, 经过酶切、连接、PCR鉴定和测序鉴定克隆到原核表达载体pCold TF中, 并转化大肠杆菌BL21(DE3), 进行IPTG诱导表达和表达产物的SDS-PAGE、Western-blotting 分析、纯化以及反应原性的iELISA检测。结果表明, 成功人工合成了长831bp的GCRV VP7蛋白基因编码框和构建了VP7蛋白基因的重组质粒pCold TF-VP7, pCold TF-VP7转化菌经IPTG(1mmol/L)诱导3—4h即可获得特异性蛋白VP7重组蛋白的高效表达, VP7重组蛋白相对分子量大小约为73.9ku, 与预期大小相符, 且与鼠抗GCRV血清有良好反应原性。
关键词:  草鱼出血病病毒  VP7  搭桥PCR  原核表达
DOI:10.11693/hyhz201303016016
分类号:
基金项目:教育部《长江学者和创新团队发展计划》创新团队项目, IRT0848 号; 四川农业大学双支计划, 00270401 号
ARTIFICIAL SYNTHESIS AND PROKARYOTIC EXPRESSION OF GRASS CARP REOVIRUS VP7 PROTEIN GENE
WEI Xian-Chao1,2, YANG Ze-Xiao1,3, WANG Yin1, PEI La-Mei1, YAO Xue-Ping1, WANG Kai-Yu1, YANG Shui-Xian1
1.College of Veterinary Medicine, Sichuan Agricultural University;2.Aquatic Technology Vocational College of Sichuan Province;3.Key laboratory of Animal Disease and Human Health of Sichuan Province
Abstract:
2 specific primers and 19 overlapping oligo primers were designed according to the gene sequences of GCRV VP7 protein published in GenBank. A 831bp DNA fragments of GCRV VP7 protein ORF with 3 mutation bases was synthesized in vitro using overlap extension PCR, and cloned into prokaryotic expression vector pCold TF through BamHⅠ-digestion, DNALigase linking, PCR identification and DNA sequencing, then the recombinant plasmid were transformed into competent Escherichia coli BL21 (DE3) for expression induced by 1mmol/L IPTG, the expression products were ana-lyzed by SDS-PAGE and Western-blotting, purified and its reactionogenicity was detected using iELISA. The results showed that the recombinant expression plasmid pCold TF-VP7 was constructed successfully, which could produce high level expression inducing with IPTG in 3—4h. The expressed fusion protein named VP7 fusion protein was about 73.9ku, consistent with the predicted size, and could be recognized by anti-GCRV serum (mouse).
Key words:  Grass Carp Hemorrhage Virus  VP7  Overlap extension PCR  Prokaryotic expression
Copyright ©  Editorial Office for Oceanologia et Limnologia Sinica    Copyright©2008 All Rights Reserved
Supervised by: China Association for Science and Technology   Sponsored by: Chinese Society for Oceanology and Limnology, Institute of Oceanology and Limnology, CAS.
Address: 7 Nanhai Road, Qingdao, China.    Postcode: 266071    Tel: 0532-82898753  E-mail: liuxiujuan@qdio.ac.cn  
Technical support: Beijing E-Tiller Co.,Ltd.