Farahmand Leila, Majidzadeh-A Keivan, Sepehrizadeh Zargham, Mofid Mohammad Reza, Esmaeili Rezvan, Yazdi Mojtaba Tabatabaei
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran ; Iranian Center for Breast Cancer (ICBC), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.
Avicenna J Med Biotechnol. 2012 Jan;4(1):15-22.
In recent years, recombinant monoclonal antibodies and their derivatives have emerged as important targeted therapy agents. Monoclonal antibodies are extremely difficult to produce. So, the cost of production is very high and many people cannot afford these drugs. In this regard, choosing inexpensive and easy ways to manipulate production systems such as bacterial hosts to reduce the cost of manufacturing these critical components are considered as vital step for developmental issues in recombinant expression systems. We, therefore, attempted to generate a polycistronic construct of anti HER-2 F(ab')2 fragment antibody for insertion in an expression bacterial plasmid. Also some modifications were made in the hinge region to express antibody F(ab')2 fragment in its authentic form preventing from multiple varieties of disulfide bond formation. Finally, synthesized construct was cloned in pET-32 Ek/LIC vector without using restriction enzyme digestion or ligation reactions. The results of this study showed that modified F(ab')2 fragment was simply and successfully inserted in Escherichia coli (E.coli) using the Ligation Independent Cloning technology.
近年来，重组单克隆抗体及其衍生物已成为重要的靶向治疗药物。单克隆抗体极难生产。因此，生产成本非常高，许多人买不起这些药物。在这方面，选择廉价且易于操作的生产系统，如细菌宿主，以降低制造这些关键组件的成本，被认为是重组表达系统发展问题的关键一步。因此，我们试图构建一种抗HER-2 F(ab')2片段抗体的多顺反子构建体，用于插入表达细菌质粒中。此外，还对铰链区进行了一些修饰，以表达真实形式的抗体F(ab')2片段，防止形成多种二硫键。最后，合成的构建体被克隆到pET-32 Ek/LIC载体中，无需使用限制性酶切或连接反应。本研究结果表明，使用不依赖连接的克隆技术，修饰后的F(ab')2片段可简单且成功地插入大肠杆菌（E.coli）中。
