The Antibacterial Activity And Their Application Of Hemoglobin And Its Peptides From Blood Clam Tegillarca Granosa

The laboratory got three genes of hemoglobin subunit of the blood clam Tegillarca granosa according to thecloning techniques, and preliminary proofs showed that the hemoglobin was involved in the antimicrobialimmune response were detected based on the quantity of mRNA expression, this study intended to verify whetherthe hemoglobin protein possess antibacterial activity based on protein experiment, and discuss the antibacterialfunction and mechanism of hemoglobin in the further research.The experimental material was blood clam reared in the aquaculture basement. Gel exclusionchromatography was adopted to separate and purify the hemoglobin, and three kinds of hemoglobins wereobtained. According to the standard curve of the molecular weight under natural state of the detected protein, thesethree substances were specuLated to be, four heterologous polymers and homologous dimers respectively. TheresuLt of Tricine-SDS-PAGE electrophoresis further prove that two kinds of hemoglobins have been separatedand purified.Digest the hemoglobin by trypsin, and get separated and purified peptides by C-18reverse phasecolumn. Agarose radial diffusion method was conducted to test the antibacterial activity of hemoglobin and itsdigestion peptides on Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Bacillus firmus, Vibrioparahaemolyticus and Vibrio alginolyticus. No antimicrobial activity was detected for hemoglobin onStaphylococcus aureus and three kinds of Vibrio, while antibacterial activity was detected for hemoglobindigestion peptides on Vibrio harveyi, Vibrio parahaemolyticus and Vibrio alginolyticus only. In addition,minimum inhibitory concentration (MIC value) of hemoglobin samples and hemoglobin digestion peptides wasmeasured by adopting96well-trace of times dilution method.The above study shows that blood clam hemoglobin and its peptides both possess varying degrees of in vitroantibacterial activity, and these resuLts verified the additional biological functions other than oxygen-carrying ininvertebrates, particuLarly in shellfish. This research contribute to expanding the understanding of the innateimmune mechanisms of shellfish and lay foundation for the further exploration of the nonspecific immunityfunction of hemoglobin.