【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。更多还原

【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 BamH Ⅰ-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 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).更多还原