Preparation Of Monoclonal Antibody Against Recombinant Ganoderma Lucidum Immunoregulatory Protein (rLZ-8) And Identification

LZ-8 (Lucidium-Lz-8) was first isolated from the fruiting bodies of Ganoderma lucidum , a fungal immunom- odulatory protein, in 1989 by kino. Monomer total of 110 amino acids ,N terminal 13 amino acids com- pose anα-helix structure and a "sandwich" lamellae composed by 9β-sheet structure in C terminal , singleα-helix of LZ-8 in N-side nested with each other form an active dimer. Previous studies have shown that fungal immunomodulatory proteins can activate the expression of several cytokines in mouse spleen cells such as IL-2, IL-4, TNF , IFN and promote cell division , because of that fungal immunomodulatory prot- eins possess immune regulation function. Reduction or complete inhibition systemic immune rejection induced by BSA (bovine serum albumin), and Fips can also act as immunosuppressive agents; in vivo th- ese proteins can prevent systemic anaphylactic reactions and significantly decrease footpad edema during the Arthus reaction(a local allergic reacti- on) , in addition to some studies have found that LZ -8 have a killing effe- ct on cancer cells.Currently , the further study of effect mechanism for LZ-8 ,a fungal immunomodulatory protein are subjected by the extremely less understand of protein receptor and active sie . antibodies and antigen-specific binding is mainly characteristics of monoclonal Antibody . By use of this characteristic, investigator can indirectly capture the receptor protein or make the antibodies as probes marker protein in the cells, though analysis the role of active sites and receptor of protein will have significationtly impact for research of protein active mechanism at the molecular and organ levelsThe gene sequence of LZ-8 are regulated according to yeast codon favor. The new synthesis gene sequence integration into the yeast genome ,after expression and purify obtain Recombinant Ganoderma Lucidum Immunore- gulatory Protein ( LZ-8 ) . Through microfiltration, ultrafiltration columns crude purify and the Sp fast flow cation exchange chromatography and the G75 molecular sieve chromatography, the high purity LZ-8 protein was obtained. Later, LZ-8 as the antigen repeatedly immunized different parts of the mice and product anti-LZ-8-specific antibodies. Take spleen cells of BALB / c mouse ,have been immuned by LZ-8 fusion with SP2 / 0 myeloma cell and application of ELISA screening.Eventually be able to retain two stable hybridoma cell lines , secreted monoclonal Antibody against Recombinant Ganoderma Lucidum Imm- unoregulatory Protein(LZ-8)and numbered clone13-2- 3 , clone11- 4-4.Following these, monoclonal antibody subtype identification, titer assay and stability analysis confirm that clone13-2 -3 cell lines serect IgG1 subclass and clone11-4-4 cell lines serect IgG2a subclass , the culture supernatant of ELISA titer are 1:3000 and 1:2800 respectively for clone13-2 -3 and clone11-4-4 hybridoma cells. After Continue culture 15 generations, the two hybridoma cell lines still have the same antibodies titer as the first generation. The lab use LZ-8 protein as antigen to obtain monoclonal antibodies in mice at first and the success obtain of LZ-8 monoclonal antibodies will pave the way done for the later research of protein active mechanism.