Isolation of T-Cell Antigens by Retrovirus-Mediated Expression Cloning
Expression cloning is a powerful tool in isolating a cDNA for a protein when the protein is difficult to purify but can be detected by antibody staining or some biological activity, such as growth promoting activity. In this chapter, we focus on the cloning of cDNAs for surface proteins by expression cloning. Originally, expression cloning was performed in Escherichia coli (E. coli) transduced with cDNA libraries constructed in lamda phage-based vectors followed by screening using antibody staining. However, proteins produced in E. coli have no glycosylation, which is a disadvantage in detecting the expression of surface proteins by antibodies because significant numbers of monoclonal antibodies can only recognize glycosylated molecules, and sometimes proteins are not properly folded in E. coli . Therefore it was recommended to use polyclonal antibodies for the expression cloning using E. coli . Later, an expression-cloning method using mammalian cells was developed; plasmid vectors harboring the SV40 replication origin are amplified in COS cells expressing the SV40 large T antigens (1 ,2 ) such that cDNA products are detected and isolated efficiently. That is, the SV40 origin-bearing plasmids can replicate in COS cells (2 ), allowing the plasmids to be recovered by transformation into E. coli . Amplification of the plasmid also greatly increases expression of the cDNA in the plasmid.
- 抗体标签制备果蝇多线染色体压片
- 沉淀反应(Precipitation reaction)
- 人碳酸酐酶(CA)酶酶联免疫分析(ELISA)
- 特异性抗原诱导的细胞毒性T细胞功能测定
- Studies of Enzymes That Cause Resistance to Aminoglycosides Antibiotics
- Purification of C1q Receptors and Functional Analysis
- Primary Ear Fibroblast Derivation from Mice
- Mast Cell Activation by Lipoproteins
- Immortalization of Human B Cells: Analysis of B Cell Repertoire and Production of Human Monoclonal Antibodies
- Heat-Induced Antigen Retrieval for Immunohistochemical Reactions in Routinely Processed Paraffin Sections