Dem'ianova N G, Bolotin A P, Novikov A A, Sorokin A V, Lebedev A N
Antibiot Med Biotekhnol. 1987 Nov;32(11):820-4.
The chemically synthesized structure gene of human proinsulin was cloned in E. coli on the secretory vector containing regulatory elements of the Bacillus amyloliquefaciens alpha-amylase gene. The proinsulin gene was inserted by the EcoRI site located immediately after the DNA area encoding the alpha-amylase signal peptide. The E. coli cells transformed by such a plasmid produced hybrid protein consisting of the alpha-amylase signal peptide, five amino acid residues after the gene mating and human proinsulin. For accurate mating of the alpha-amylase gene leader sequence and proinsulin gene directed mutagenesis was performed on the filiform phage M13 mp9 with synthetic oligonucleotide. The hybrid gene was transferred to the vector molecule capable of replicating in Bacillus subtilis. It was shown that in the cells of both E. coli and B. subtilis there is synthesized protein interacting by the radio-immunological data with antibodies to porcine insulin, a large portion of immunologically active protein being detected in the periplasmic space of E. coli cells and in the culture fluid of B. subtilis cells which was indicative of proinsulin secretion directed by the alpha-amilase regulatory elements.
人胰岛素原的化学合成结构基因被克隆到大肠杆菌中，该大肠杆菌搭载了含有解淀粉芽孢杆菌α-淀粉酶基因调控元件的分泌载体。胰岛素原基因通过位于编码α-淀粉酶信号肽的DNA区域之后的EcoRI位点插入。用这种质粒转化的大肠杆菌细胞产生了由α-淀粉酶信号肽、基因融合后的五个氨基酸残基和人胰岛素原组成的杂合蛋白。为了使α-淀粉酶基因前导序列与胰岛素原基因精确融合，对丝状噬菌体M13 mp9进行了合成寡核苷酸定向诱变。杂合基因被转移到能够在枯草芽孢杆菌中复制的载体分子上。结果表明，根据放射免疫数据，在大肠杆菌和枯草芽孢杆菌的细胞中都合成了能与抗猪胰岛素抗体相互作用的蛋白质，在大肠杆菌细胞的周质空间和枯草芽孢杆菌细胞的培养液中检测到了大部分具有免疫活性的蛋白质，这表明α-淀粉酶调控元件指导了胰岛素原的分泌。
