Solube Express Of SEB And Construct A SEB Rabbit Single-chain Fv Library

The study of staphylococcal enterotoxin B (SEB ) have never been stopped since it was discovered in 1954, SEB is a common cause of food poisoning and toxic shock. SEB can make Rhesus monkeys vomit, this mechanism is relate to stimulation of vomit nerve centre of pneumogastric nerve and sympathetic nerve,it also can conduce diarrhea,excessive and frequent evacuation of watery feces, usually indicating gastrointestinal distress or disorder,even shock and death. SEB is also an important member of the superantigen family, which exerts a number of pathological effects in the human, as well as susceptible animals. With the development of anti-tumor medicines, more attention has focused on it. As a superantigen, SEB possesses the ability to bind to major histocompatibility complex class II molecules and be recognized by T cells bearing certain T-cell receptor (TCR) V beta alleles. Superantigen-mediated T cell activation requires the participation of antigen-presenting cells (APC). Once superantigen has bound class II MHC molecules on the surface of APC, it then can interact with the T cell receptor to induce T cell activation. Superantigen-mediated T lymphocyte activation, along with its consequent cytokine production is thought to be the basis for the pathophysiology of conditions such as toxic shock syndrome and possibly rheumatoid arthritis. SEB can activate T-lymphocytes, include the release of lymphokine and cause the lysis of tumor cells. The output of SEB from native staphylococcal is low and it is hard to purify. In this study, we want to clone SEB gene and express soluble SEB in E.coli. At this time there are no vaccines or therapeutics to protect against SEB exposure. We also want to construct a single-chain fragment (ScFv) library in which in order to select antibody binding specifically to SEB. The gene of SEB is amplifyied with PCR from SEB producing strain 1169 and is cloned into expression vector pTIG-Trx for soluble express in E.coli.The crude expressed protein was identified by SDS-PAGE and Western, then the product was purified by Probond resin. We use the pure SEB immunize rabbit,extact total RNA from spleen, by means of phage display technology we construct combinatorial immunoglobulin library. The gene of SEB is obtained by PCR, and get soluable expression in the host strain BL21 (DE3) , the last purifyied production is about 150mg/l. By means of phage display we construct a library, capacity of this library is about 1011, it can be used in the next processs.