Rapid immunosorting of transmembrane proteins of Lymphocytes from a cDNA expression library of COS-1 cells.
The proteins on lymphocyte surface play important roles in a wide range of immunological processes, but the profile and characterization of surface proteins remain to be further investigated, among which the method for fast screening of surface proteins needs to be established. In this study, a conventional cDNA clone library of hepatic lymphocytes from C57BL/6 mouse was constructed, and then the cDNA was inserted into a recombinant expression vector pSecTag-attR with a signal peptide and tag protein for fluorescence screening. The recombinant cDNA expression library was transfected into COS-1 cells, and the transfected cells with the expressed membrane proteins were labeled by fluorescence antibodies and isolated by fluorescence activated cell sorting. After two cycles of sorting, the purity of fluorescence positive cells with membrane proteins was up to 98%, and the representative membrane molecules on lymphocytes such as CD3, CD4, CD8, NK1.1 and NKG2D were detected in the library. These results demonstrated that the cDNA expression library containing transmembrane proteins provided an efficient and fast tool for the study of transmembrane proteins on hepatic lymphocytes.
Ge
Kuikui
K
Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China.
Sun
Rui
R
Tian
Zhigang
Z
D000818:Animals; D000950:Antigens, Ly / Q000235:genetics / Q000302:isolation & purification; D017252:CD3 Complex / Q000235:genetics / Q000302:isolation & purification; D015704:CD4 Antigens / Q000235:genetics / Q000302:isolation & purification; D016827:CD8 Antigens / Q000235:genetics / Q000302:isolation & purification; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D008214:Lymphocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D055651:NK Cell Lectin-Like Receptor Subfamily B / Q000235:genetics / Q000302:isolation & purification; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D000950:Antigens, Ly; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; C527211:Klrb1c protein, mouse; C527086:Klrk1 protein, mouse; D008565:Membrane Proteins; D055651:NK Cell Lectin-Like Receptor Subfamily B; D055655:NK Cell Lectin-Like Receptor Subfamily K