Prokaryotic Expression Polyclonal Antibody Preparation And Purification Of Abalone Ferritin And Hemocyanin

Ferritin and hemocyanin are multifunctional proteins which both contain metal ions, they directly or indirectly involve in detoxification of heavy metal ions in vivo role and participate in a variety of physiological and biochemical responses of organisms and a variety of metal proteins and enzymes synthesis. When pathogenic infects the organism, the expression of ferritin and hemocyanin in the organism have upward trend, it's obvious that ferritin and hemocyanin have closely relationship with the immune.Ferritin composing of protein shell and ferric-oxyhydroxy phosphate core has a unique cage-like structure that similar to spherical virus, its basic function is storing and releasing ions in biological vivo, regulating intracellular iron concentration and keeping iron metabolic balance in the cells, in order to prevent iron poisoning. Abalone iron protein has two different polypeptide subunits, namely Abalone ferritin subunit 1(AbFe1) and Abalone ferritin subunit 2 (AbFe2), which is similar to vertebrate ferritin.Hemocyanin is the second largest oxygen carrier protein of animal, mainly located in gastropod mollusks, cephalopods and crustaceans and other arthropods blood lymphocytes. Abalone hemocyanin (AbHc) formed by the 20 polymers in the concave cylindrical structure has two different subunits, AbHc1 and AbHc2, each subunit has eight Functional units (FUs), named as a ~ h, its molecular weight is about 400KDa, this structure is similar to single-hole snail hemocyanin. The previous researches show that, the homology of amino acid sequence among a ~ h functional body is over 45 percent, different functional bodies may evolve by the same protein molecule.This study selected the ORF sequences in two subunit (AbFe1 AbFe2) of abalone ferritin and part of the sequence AbHc-d and AbHc-h in FU-d, FU-h functional unit of AbHcH1 subunit in abalone hemocyanin to put up Prokaryotic expression, it used Freund's adjuvant to emulsify target protein and took the method of multi-point injected the muscles of immunized rabbits; after 4 to 5 times immunization, it gained four antiserum, namely as anti-AbFe1, AbFe2, AbHc1d and AbHc1h. Western blot analysis revealed that anti AbFe1, AbHc1d recombinant protein have lower antibody titers, and the specificity of anti AbFe2 and AbHc1h were better, AbFe1 antibodies can recognized AbFe2 protein; besides the antibodies of AbHc1h and AbHc1d can identified the protein of AbHc1h and AbHc1d. To further verify the specificities of antibodies'identification, this research used 50% and 33% saturated ammonium sulfate to fractionate and purify antiserum, used acetone extracts of the host bacteria to remove the background induced by cross-reactive between antibodies and the host strain. After Western blot, the background of antibodies that purified by saturated ammonium sulphate reduced, the loss of the antibodies decreased, nevertheless when used acetone extracts of the host bacteria to remove the background, the antibody titers significantly reduced. Meanwhile, in order to find the best working concentration of antibodies and antigen combine and study the specificities of antibodies'identification among the protein subunits, it used electroeluting method to recover expressed target protein and enzyme-linked immunosorbent assay (ELISA) to screen the best combine concentration of antiserum and antigen of AbHc1d and AbHc1h. The results revealed that the best dilute multiple was 1000, the concentration of antigen were 4μg/mL, 2μg/mL. On the basis of confirming the best working concentration of antibodies and antigen combine, competitive ELISA test of AbHc1d and AbHc1h versus each antibody was conducted in order to detect the specificity of antibodies and the extent of non-specific recognition.Through preparing AbFe1, AbFe2 of ferritin subunits and polyclonal antibody of FU-d, FU-h functional body of AbHcH1 subunit in hemocyanin, this study detected specific recognition among the protein polymer subunits, it had done some preliminary work in order to further verify whether ferritin and hemocyanin polymer structure isomerizes, when pathogen infects abalone host.