Purification And Characterization Of α₂-macroglobulin From Grass Carp Ctenopharyngodon Idellus And Cloning Of Part Of Its Gene

2-Macroglobulin is one of the non-specific protease-inhibitor existed in many animals, including both vertebrates and invertebrates. It plays an important role in the resistance against invasion of pathogenic microbe. Aeromonas hydrophila (Ah) was considered as a major pathogen for cultured fishes, against which grass carp had a natural resistance, 2-Macroglobulin was purified from grass carp plasma by precipitation with 5%-18% polyethylene glycol (PEG) 6000, and dialyzed in 50 mmol/L Tris-HCl (pH 7.4) buffer. As a result the grass carp 2M crude product was obtained. The crude product passed through High SepHacryls200 high Resolusion gel filtration chromatography column and the part with activity was collected and concentrated. Furthermore the product was through Hiprep l6/10QXL anion-exchange chromatography column. The three steps of the procedure resulted in the purification of grass carp plasma 2M. The purified product was analyzed through polyacrylamide gel electrophoresis (PAGE) under natural condition and the protein showed a single band. Meanwhile, it was analyzed through SDS-PAGE under reducing conditions and the proteins showed double bands with molecular weight of about 95kD and 80kD respectively. It demonstrated that grass carp a 2M was composed of two distinct subunits.Most properties of grass carp 2M were similar to those of human 2M. Grass carp 2M treated with trypsin produced fast-form of the molecule mobile faster in PAGE, but the untreated grass carp 2M had the property of electrophoretical slow-form, 2M was a nonspecific proteinase inhibitors in blood plasma. Inhibition of activity of Aeromonase hydrophilas extracellular proteinase (AhECPase) showed that grass carp 2M could inhibit the proteinases secreted from invading bacteria. Double unmudiffusion of 2M demonstrated no cross-antigenicity between grass carp's and human 2M.Total RNA was isolated from liver tissue of common carp (Cyprinus carpio) and grasscarp (Ctenopharyngodon idellus). Two pairs of primers were designed based on analysis of conservative region and bait region sequences of common carp α2M gene. Using RT-PCR method, conservative region sequencess of α2M were respectively amplified and cloned and the sequences including bait region of a2M gene was amplified and cloned from total RNA which was extracted from the grass carp liver tissue. The cloned cDNA was sequenced and the corresponding amino acid sequence of the bait region of grass carp α2M was predicted. Analysis of amino acid sequence indicated that cloned sequence of grass carp α2M was distinctly different from those of the common carp in the bait region of α2M. The difference among various animals' α2M in the function of inhibition activity of extracellular proteinase secreted from invading bacteria had close relationship with the one among the bait region.Aeromonas hydrophila J-l strain was incubated into LB medium, and cultured at 28 C for 24 hours. The bacteria was collected through centrifugation and inactivated by 90 C 30min in order to get whole bacterina. In addition, the supernatant of medium was processed through the following procedures: firstly it was precipitated with 20%-60% (NH4) 2SO4 solution, then the part was inactivated by 90 C 30min with the purpose of obtaining the toxin vaccine. Proper dose grass carp a 2M were respectively mixed with the two vaccines mentioned above. Five kinds of feeds were manufactured containing the whole bacterina, the toxin vaccine, the whole bacterium vaccine with a 2M ,the toxin vaccine with a 2M and the common feed as the control respectively. The experiment fishes were divided into five groups, with 30 /group. The five kinds of feeds were fed to experiment fishes respectively for successive 15 days and the common feed for further successive 20 days. Meanwhile, the control group was fed with the common feed for 35 days. All five groups were incubated with AhJ-1 strain 50LD50, The results were as following: the whole bacterium vaccine had the relative percentage survival(RPS) of 43.3%, the whole bacterium,...