Purification Of A Protein From Coelomic Fluid Of The Earthworm Eisenia Foetida And The Estimation Of Its Biological Activities

The coelomic fluid of earthworm Eisenia foetida contains various biological active proteins. Up to now, several kinds of biological active proteins have been identified in the coelomic fluid of earthworm, and their antibacterial activity and hemolytic activity were primarily estimated. However, the earthworm proteins from coelomic fluid are rich in species numbers, and we have a little of understanding for earthworm proteins from coelomic fluid. We selected Eisenia foetida as experimental material, and a novel protein from coelomic fluid was isolated and purify, and its physicochemical index and biological activity were further studied.We used ultrafiltration, gel chromatography and ion exchange chromatography methods to separate a novel protein, named as Eisenia foetida Coelomic Fluid Protein (ECFP). ECFP was characterized by SDS-PAGE with molecular weight of 38.6 kDa. Ultraviolet analysis indicated ECFP's maximum absorption wavelength of 278 nm, and ion exchange chromatography determined ECFP to be a cationic protein. Average concentration of ECFP was 0.349 mg/mL and average pH value was 7.95 in 10 samples. Ninhydrin reaction, biuret reaction and 5-Sulfosalicylic acid reaction all showed positive.Several biological activities for ECFP were studied as follows:1. Turbidimetry and Oxford cup method was performed to determine the inhibition effect of ECFP on Escherichia coli and Staphylococcus aureus. The results of turbidimetry showed that ECFP displayed the inhibition effects on the growth of Escherichia coli in 1 h and Staphylococcus aureus in 3 h. Oxford cup test showed that ECFP displayed the inhibition zone in Staphylococcus aureus. Inhibiting diameter were 13.2 mm, 12.2 mm and 11.0 mm respectively when bacteria were treated with the final ECFP concentration of 0.32 mg/mL, 0.16 mg/mL and 0.08 mg/mL ECFP in 24 h, but inhibition zone of Escherichia coli was not observed in 24 h.2. Hemolytic activity on chicken erythrocytes was detected with different concentration of ECFP. The test results showed that ECFP exhibited significant lysis effects on chicken erythrocytes, and the minimum effect concentration was 0.19μg/mL in 2 h. When chicken erythrocytes were treated with high concentration of ECFP, cell dissolution was observed under inverted microscope.3. MTT test was performed to determine the inhibition effect of ECFP on Hela cells and LTEP-α-2 cells. The results showed that incubation from 24 to 96 h with the final ECFP concentrations of 0.16, 0.08, 0.04, 0.02 and 0.01 mg/mL respectively. ECFP had increased gradually positive-inhibiting effect on the Hela cells and the LTEP-α-2 cells. Moreover, the inhibition ratio significantly increased from 24 to 96 h when the concentration of ECFP was 0.16 mg/mL, and ECFP at other concentrations had the same results on the Hela cells and the LTEP-α-2 cells (P<0.01) in a dose-dependent manner. The inhibition ratio increased significantly at the concentration of ECFP from 0.01 to 0.16 mg/mL in 24 h, and others time points (48,72,96 h) had the same results on the Hela cells and the LTEP-α-2 cells (P<0.05) in a time-dependent manner. The morphological changes of Hela cells and LTEP-α-2 cells treated with different concentration of ECFP in 72 h were characterized with decreasing attachment rate, plasmatorrhexis and necrosis after treatment for 72 h at the concentration of ECFP from 0.01 to 0.16 mg/mL. The LTEP-α-2 cells were characterized by decreasing attachment rate, vacuole-shape in the cytoplasm and necrosis after treatment for 72 h at the concentration of ECFP from 0.01 to 0.16 mg/mL. 4. White mice were intraperitoneal injection of ECFP whose final concentrations were 0.16, 0.08, 0.04, 0.02 and 0.01 mg/mL respectively, and the body temperature was detected from 0 to 96 h. The results indicated that the body temperature of white mice didn't increase significantly from 0 to 96 h when the concentration of ECFP was 0.16 mg/mL, and the same results (P>0.05) were determined at other concentrations of ECFP. The results indicated that the white mice body temperature didn't increase significantly at the concentration of ECFP from 0.01 to 0.16 mg/mL in 96 h, and the same results (P>0.05) was found at others time point (0, 24, 48, 72 h). White mice didn't occurred adverse reaction, as well as anorexia and spirit, which indicated that ECFP had no obvious influence on the body temperature of white mice.Furthermore, after the biological activities of ECFP were confirmed, we used orthogonal test to successfully separate and purify the protein extract from earthworm homogenate liquid. Studies on its activities resulted in a bacteriostatic activities on Staphylococcus aureus, which indicated earthworm homogenate liquid might also contain antibacterial protein.