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目的 在原核细胞表达抗HIV-1gp120单链抗体基因,纯化后检测其活性。方法 将单链抗体基因插入pET28原核表达载体进行诱导表达,对包涵体进行变性复性处理后,利用Ni-NTA金属螯合层析柱对表达的蛋白进行初步纯化,纯化后的单链抗体与gp120标准抗原膜条反应。结果 表达产物主要以包涵体形式存在,最高表达量占菌体总蛋白量的51％,金属螯合层析一步纯化后纯度超过85％。纯化后的单链抗体可以特异地识别gp120标准抗原。结论 在原核细胞表达的单链抗体,复性纯化后具有生物学活性。
Objective To express anti-HIV-1gp120 single-chain antibody gene in prokaryotic cells and determine its activity after purification. Methods The single-chain antibody gene was inserted into the prokaryotic expression vector pET28 to induce expression. The inclusion body was denatured and renaturated, and then the expressed protein was purified by Ni-NTA metal chelate chromatography. The purified single-chain antibody gp120 standard antigen membrane reaction. Results The expressed product mainly existed in the form of inclusion body. The highest expression level accounted for 51% of the total amount of the bacterial cells. The purity of metal chelate chromatography was over 85% after one-step purification. The purified single chain antibody can specifically recognize the gp120 standard antigen. Conclusion The single chain antibody expressed in prokaryotic cells has the biological activity after refolding and purifying.