Isolation And Purification Of Antibacterial Polypeptide From Cow Uterine Endometrium

Separation and purification of antimicrobial peptides from cow uterus mucus and cow uterus tissue have not been reported at home and abroad. Cow uterus mucus and cow uterus tissue are regarded as study objects to extract and purify cow uterus mucus and cow uterus tissue antimicrobial peptides, and antibacterial activity of level electroelution elution sample and high-performance liquid chromatography (RP-HPLC) separation sample were detected by agarose diffusion method ,respectively.Here are two parts in this article.Partâ… Separation, purification and analysis of antibacterial activity of Cow uterine mucus antimicrobial peptide.Fresh cow uterine tissue extracted by 5% acetic acid, the antimicrobial activity of the acid-soluble extracts of cow uterine mucus were assayed by gel overlay technique,the corresponding antimicrobial protein bands were cut out from the gel of prepared acid-urea polyacrylamide gel electrophoresis(AU-PAGE), and the proteins in the gel was eluted by electrophoresis. The elution proteins were dialyzed with distill water, and Iyophilized. And an antibacterial band was found when the elution sample of the antimicrobial protein was detected by agarose gel diffusion method. The elution sample of the antimicrobial protein was purified by RP-HPLCA molecule at 45 minute of retention time could effectively kill E.coli BL21 and Staphylococcus aureus 26003 , and the molecular weight of the molecule determined by Tricine-SDS-PAG electrophoresis is about 14 kD.Partâ…¡Separation,purification and analysis of antibacterial activity of cow uterine tissue antimicrobial peptide.Fresh cow uterine tissue extracted by 5% acetic acid, the antimicrobial activity of the acid-soluble extracts of cow uterine mucus and tissue were assayed by gel overlay technique,the corresponding antimicrobial protein bands were cut out from the gel of prepared acid-urea polyacrylamide gel electrophoresis(AU-PAGE), and the proteins in the gel was eluted by electrophoresis. The elution proteins were dialyzed with distill water, and lyophilized. Two antibacterial bands which were against E. coli BL-21, Staphylococcus aureus 26003, E. coli 4605, Pseudomonas aeruginosa 10104. were found when the elution sample of the uterine antimicrobial protein was detected by agarose gel electrophoresis diffusion. The above antimicrobial protein of the elution sample was purified by RP-HPLC. A molecule at 45 minute of retention time which could efectively kill Ecoli BL21was detected. While single molecule at 35 minute,43minute and 45minute of retention time could efectively kill Ecoli BL21 determined by agarose radial diffusion assay.The antimicrobial peptides of molecular weight was determined by Tricine-SDS-PAG electrophoresis. The results showed that the above antimicrobial protein which is about 14kD from cow uterine tissue is similar to the antimicrobial protein from cow uterine mucus, demonstrating they may be the same material; while the molecular weight of the below antimicrobial protein purified by RP-HPLC at 35 minute,43minute and 45minute were about 14-16kD, 6kD and 12kD the antimicrobial peptide,respectively.In a word, electrophoresis and RP-HPLC technique was first applied to isolate antimicrobial peptide from the cow uterus mucus and tissues, and an analysis of its molecular size and antibacterial activity were carrid out, which lay foundation for further researched into molecular mechanism of cow uterus endometrial antimicrobial peptides and construction of efficient prokaryotic, eukaryotic expression vector in future ,and will provide necessary information for developing the antimicrobial peptides of cows endometrial into new generation anti-infective drugs .