Apal'ko S V, Lunin V G, Filipenko M L, Matveeva V A, Liashchuk A M, Lavrova N V, Sherina E A, Aver'ianov A V, Kostianko M V, Glushkov A N
Mol Gen Mikrobiol Virusol. 2011(2):24-30.
Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.
构建了两种重组质粒。第一种质粒包含由苯并[a]芘免疫模拟肽、噬菌体M13的pIII蛋白和纤维素结合结构域编码序列组成的杂合基因。第二种质粒包含由噬菌体M13的pIII蛋白信号肽、苯并[a]芘免疫模拟肽、噬菌体M13的pIII蛋白和纤维素结合结构域序列组成的杂合基因。所获得的重组质粒用于基于大肠杆菌M15菌株表达含苯并[a]芘免疫模拟肽的嵌合蛋白。结果表明使用第一种质粒时重组蛋白不表达。同时，结果显示存在含噬菌体pIII蛋白信号肽的免疫模拟肽的重组蛋白积累。这种嵌合蛋白以“成熟”（无信号肽）和“未加工”（有信号肽）形式产生。使用蛋白质免疫印迹分析表明，只有“成熟”形式特异性结合抗苯并[a]芘的B2单克隆抗体。因此，我们表达、纯化并鉴定了含苯并[a]芘免疫模拟肽的重组蛋白。
