白喉毒素DAB389-aMSH重组毒素的构建及表达

Construction and Expression of Recombinant Toxin DAB389-aMSH

关键词:  DAB389-aMSH  重组毒素  构建  表达

Key words:  DAB389-aMSH  recombinant protein  construction  expression  

摘要:

摘要:利用含有白喉毒素N端序列的基因作上游引物、含有aMSH全序列作下游引物，以pET28a/DAB389-EGF为模板，PCR扩增DAB389-aMSH基因片段，用限制性内切酶EcoR I和Nco I酶切，并插入原核表达载体pET28a的相应位点，构建了重组表达载体pET28a/DAB389-aMSH，在大肠杆菌中表达重组融合蛋白DAB389 -aMSH，转化菌经1mmol/L IPTG、30C诱导5h，用SDS-PAGE 和Western blot 鉴定表达的重组毒素质。结果表明扩增的片段与理论值一致。重组质粒的DNA序列分析正确。SDS-PAGE表明，重组毒素相对分子量为43.76KD，且表达量达菌体总蛋白量的31.6%左右。Western blot分析显示，重组毒素能特异性地与抗白喉抗体结合,表明已成功构建表达了重组DAB389-aMSH的工程菌株，表达产物具有生物学活性。

Abstract:

Abstract: The DAB389 -aMSH gene fragment was amplified from plasmid pET28a/DAB389-EGF by PCR with the upper primer containing diphtheria toxin complementary sequences, and the down primer containing aMSH complementary sequences. The PCR product was digested by the restriction enzymes NcoI and EcoRI, and cloned into Nco I/EcoR I site of expression vector pET28a, resulting in plasmid pET28a/ DAB389-aMSH. The recombinant plasmid was transformed into E.coli BL21 and induced by 1mmol/L IPTG 30C 5h, It was demonstrated by SDS-PAGE and Gel scanned analysis that the recombinant toxin was expressed soluble and reached 31.6% of the total protein, the Mr is 43.76KD. So the engineering bacteria of recombinant plasmid DAB389-aMSH with biologic activity was successfully constructed.