Mahdizadeh Hossein, Salimian Jafar, Noormohammadi Zahra, Amani Jafar, Halabian Raheleh, Panahi Yunes
Department of Biology, Science and Research branch, Islamic Azad University, Tehran, Iran.
Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Iran J Pharm Res. 2020 Summer;19(3):329-348. doi: 10.22037/ijpr.2020.112959.14040.
CTLA4-Ig (Abatacept) has been produced to suppress immune response by inhibition of T cells functions in autoimmune disease. A new drug, which is called belatacept, has recently been recently developed that is more efficient. The development has been occurred by two substitutions (A29Y, L104E) in the extracellular domain of CTLA4. In the present study, the bioinformatics analysis was used in order to make a new structure that has a better function in comparison with belatacept. Firstly, eight different structures were designed. Thereafter, the secondary and 3D structures, mRNA structure, docking of chimeric proteins with CD80/CD86, antigenicity and affinity of designed chimeric molecules were predicted. Based on the criteria, a new candidate molecule was selected and its gene synthesized. The gene was cloned and expressed in BL21 (DE3) successfully. The purified rCTLA4-Ig was analyzed by SDS-PAGE, western blotting, and ELISA. Circular dichroism analysis (CD analysis) was used for characterization of the rCTLA4-Ig. Affinity of rCTLA4-Ig was also evaluated by the flow cytometry method. Finally, its biological activity was determined by T cell inhibition test. The results showed rCTLA4-Ig and the belatacept protein have some similarities in structure and function. In addition, rCTLA4-Ig was able to bind CD80/CD86 and inhibit T cell function. Although flow cytomery results showed that the standard protein (CTLA4-Ig), represented better affinity than rCTLA4-Ig, the recombinant protein was able to inhibit T cell proliferation as well as CTLA4-Ig.
CTLA4-Ig（阿巴西普）已被制备用于通过抑制自身免疫性疾病中T细胞的功能来抑制免疫反应。最近开发了一种名为贝拉西普的新药，其效率更高。这种开发是通过CTLA4细胞外结构域中的两个取代（A29Y、L104E）实现的。在本研究中，使用生物信息学分析来构建一种与贝拉西普相比具有更好功能的新结构。首先，设计了八种不同的结构。此后，预测了二级和三维结构、mRNA结构、嵌合蛋白与CD80/CD86的对接、设计的嵌合分子的抗原性和亲和力。根据这些标准，选择了一种新的候选分子并合成了其基因。该基因成功克隆并在BL21（DE3）中表达。通过SDS-PAGE、蛋白质印迹和ELISA对纯化的rCTLA4-Ig进行分析。使用圆二色性分析（CD分析）对rCTLA4-Ig进行表征。还通过流式细胞术方法评估rCTLA4-Ig的亲和力。最后，通过T细胞抑制试验确定其生物学活性。结果表明，rCTLA4-Ig与贝拉西普蛋白在结构和功能上有一些相似之处。此外，rCTLA4-Ig能够结合CD80/CD86并抑制T细胞功能。尽管流式细胞术结果表明标准蛋白（CTLA4-Ig）的亲和力比rCTLA4-Ig更好，但重组蛋白能够像CTLA4-Ig一样抑制T细胞增殖。
