摘要
根据GenBank已发表的GCRVVP7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GCRVVP7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pColdTF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,pColdTF-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。
2 specific primers and 19 overlapping oligo primers were designed according to the gene sequences of GCRV VP7 protein published in GenBank. A 83 lbp 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 BamH I -digestion, DNALigase linking, PCR identification and DNA sequencing, then the recombinant plasmid were trans- formed into competent Escherichia coli BL21 （DE3） for expression induced by lmmol/L IPTG, the expression products were analyzed 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）.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2013年第3期645-650,共6页
Oceanologia Et Limnologia Sinica
基金
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
关键词
草鱼出血病病毒
VP7
搭桥PCR
原核表达
Grass Carp Hemorrhage Virus
VP7
Overlap extension PCR
Prokaryotic expression
