摘要
利用套叠PCR技术合成重组人胰岛素基因,并在A链和B链之间加入Kex2酶切位点。将合成的重组人胰岛素基因以正确的阅读框插入到组成型分泌表达载体pGAPZα-A的α-factor信号肽序列的下游,构建成pGAPZα-A/Insulin重组分泌表达载体。表达载体用BlnI线性化处理后电击转化毕赤酵母GS115感受态细胞,构建成分泌型表达Insulin的工程菌。SDS-PAGE和Native-PAGE的分析表明:蛋白在分泌表达过程中,加入Insu-lin的A链(2.4k)和B链(3.4k)之间的Kex2酶切位点发挥了作用,Kex2蛋白酶将A链和B链切开,在α-factor信号肽的作用下通过分泌途径将A链和B链分泌到胞外,同时A链和B链又以二硫键形成了高级结构。Western Blot结果表明:重组蛋白能够与鼠抗人Insulin抗体发生特异性反应,具有与天然Insulin相同的免疫原性。
The recombinant human insulin gene was connected by overlap extension PCR. The Kex2 cleavage site was inserted between A chain and B chain. Then, the recombinant human insulin gene was ligated into pGAPZα-A vector. In order to generate a secreting expression vector pGAPZα-A/Insulin, the open reading frame of the gene was ligated with the vector of pGAPZα-A, on the downstream encoding the α-factor signal sequence. After being linearized by Bin I, the recombinant vector was transformed into competent cells of Pichia pastoris GS115 by electroporation. The analysis of SDS-PAGE and Native-PAGE showed that A chain and B chain were cleaved by Kex2 proteinase during the secretory expression of insulin. The analysis of Western blot indicated that recombinant human insulin had the antigenicity to rat anti-human insulin monoelonal antibody, which had the same immunogenicity as natural human insulin.
出处
《药物生物技术》
CAS
CSCD
2009年第2期108-112,共5页
Pharmaceutical Biotechnology
关键词
重组人胰岛素
毕赤酵母
分泌表达
Recombinant human insulin, Pichia pastoris, Secretory expression
