Document Type : Research Paper

Authors

• hossein Mahdizadeh ¹
• jafar salimian ²
• jafar amani ³
• Raheleh Halabian ³
• Zahra Noormohammadi ⁴
• Yunes Panahi ²

¹ karmand

² Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

³ Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.

⁴ Department of Biology, Science and Research branch, Islamic Azad University, Tehran, Iran

Abstract

Aim and Background: The chimer structure consisting of the extracellular domain of CTLA4 and the FC fragment of human IgG1 antibody binds to CD80 (B7-1) and CD86 (B7-2) ligands on APCs as a result inhibits CD28 binding and T lymphocyte activation. This structure, called Abatasept, was approved by the US FDA in 2005 to treat rheumatoid arthritis.



Material and methods: In this study, the CTLA4-FC chimer structure was designed and then some of its features were investigated using bioinformatics tools. The gene was then cloned into the pET-28a plasmid and transformed into E.coli BL21DE3. Recombinant protein was expressed and purified, and flow cytometry was used to evaluate the binding affinity. Mixed lymphocyte culture (MLR) method was also used to evaluate the biological activity of recombinant protein.



Results: Bioinformatics results showed that the designed structure of CTLA4-FC is suitable in terms of physicochemical properties, second and third structure, how it binds to the receptor, antigenic properties and mRNA properties. Recombinant protein was well expressed in bacteria. Purification was performed as required. Western blotting and ELISA results were positive and flow cytometry and MLR tests were acceptable.



Conclusion: Recombinant CTLA4-FC protein was produced in bacterial cells and bound well to CD80 and CD86 receptors and was able to inhibit T cells. So it can be a good candidate for drug production.

Keywords

• bioinformatics
• Abatasept
• CTLA4-FC
• flow cytometry

Main Subjects

• Biotechnology