title: Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells.
abstract: CD45RA has been found highly expressed on leukemia cells and may be a potential target of the disease. In this study, an anti-CD45RA single-chain antibody fragment (scFv3A4) was genetically linked to the N terminus of the enhanced green fluorescent protein (EGFP) to generate a scFv3A4-EGFP fusion protein. The scFv3A4-EGFP with a molecular weight of 57kDa was stably expressed and secreted from the transfected CHO cells through the ER/Golgi-dependent pathway. The fusion protein was soluble in the culture supernatant and the yield was 1350μg/L. Flow cytometry analysis showed that the scFv3A4-EGFP had the same binding site and a very similar reactivity pattern with its parental murine monoclonal antibody (mAb) 3A4. Furthermore, comparing to conventional labeled 3A4-FITC antibody, the scFv3A4-EGFP was more resistant to illumination and more suitable for immunofluorescence histology (IFH) detection. Therefore, the scFv3A4-EGFP fusion protein can be a powerful tool to investigate the targeting of CD45RA on leukemia cells, biological activity of the target and possibly for the genetic manipulation of the antibody.
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China.
authors.forename: Zhujun
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China.
authors.forename: Sisi
authors.initials: S
authors.lastname: Li
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China.
authors.forename: Yuping
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China.
authors.forename: Haizhao
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China.
authors.forename: Ming
authors.initials: M
authors.lastname: Jia
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China.
authors.forename: Zebin
authors.initials: Z
authors.lastname: Luo
authors.affiliation: Division of Hematology and Oncology, Children's Hospital of Zhejiang University School of Medicine, Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, PR China. Electronic address: Y_M_TANG@zju.edu.cn.
authors.forename: Yongmin
authors.initials: Y
authors.lastname: Tang
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D017493:Leukocyte Common Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D017493:Leukocyte Common Antigens
keywords: Anti-CD45RA antibody; CHO cell expression system; Enhanced green fluorescent protein (EGFP); Leukemia