Tati Kadijeh, Yazdanpanah-Samani Mahsa, Ramezani Amin, Mahmoudi Maymand Elham, Ghaderi Abbas
Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical sciences, Shiraz, Iran; Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical sciences, Shiraz, Iran.
Rep Biochem Mol Biol. 2016 Oct;5(1):56-61.
CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details.
Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods.
Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces.
In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids.
CD52是一种在其C末端带有糖基磷脂酰肌醇（GPI）锚定的小糖蛋白。正常和恶性的T和B淋巴细胞以及单核细胞均表达CD52。慢性淋巴细胞白血病（CLL）患者的血浆中可检测到可溶性CD52，其可用作肿瘤标志物。尽管CD52的生物学功能尚不清楚，但似乎CD52可能参与T细胞的迁移和激活。本研究的目的是在CHO细胞系中克隆并表达人CD52基因，并更详细地研究其功能。
基于GenBank数据库设计了两条特异性引物用于扩增cd52基因。从Raji细胞系中提取总RNA并合成cDNA。将扩增片段克隆到pBudCE4.1载体中。使用电穿孔法将新构建体转染到CHO-K1细胞系中。通过实时PCR和流式细胞术方法评估重组CD52蛋白的表达。
使用特异性引物对Raji cDNA进行CD52基因扩增显示出一条209 bp的条带。通过PCR、限制性酶切图谱和序列分析对新构建体进行了确认。新构建体命名为pBudKT1。RT-PCR分析在转染细胞中检测到了mRNA，流式细胞术结果显示78.4%的细胞表面表达CD52。
总之，我们建立了人CD52阳性细胞系CHO-CD52，且该蛋白在细胞膜上表达。CD52基因的克隆可能是生产治疗性单克隆抗体以及生物体液中可溶性CD52检测系统的第一步。
