Cloning And Expression Of Antimicrobial Peptides Of Cathelicidin In Bovine Marrow

Bovine Mastitis is one of the three diseases which can cause great loss in dairy industry and it is difficult for prevention and cure to the disease because long-term antibiotic therapy leads to the formation of resistants. Cathelicidin antibacterial peptides have very broad antimicrobial spectrum, which play an important role in innate immunity for mammals. In recent years, with the development of molecular biology, new peptide drugs have been developed, which hasl provide a new way for prevention and cure of mastitis.In this study, calf bone marrow was used for the material and total RNA was extracted. Indolicidin and BMAP-28 gene fragments were amplified by using RT-PCR and were cloned into pMD-18 vector. After identification by PCR, restriction enzyme digestion and sequencing analysis, the recombinant plasmid pMD18-T-Indolicidin and pMD18-T-BMAP28 were confirmed. BMAP-28 gene signal peptide was amplified by using recombinant plasmid pMD18-T-BMAP28 as a template, connected to the original expression vector pET-28a, and transformed into host strain Rosetta induced by IPTG. SDS-PAGE and Western-blot were used to detect expression product and protein was recycled. The antimicrobial activity of the target protein was tested by the major mastitis pathogens separated from a certain farm and standards strain purchased.The results showed that:1. There was one base difference was in Indolicidin gene between cloned and published. However, the amino acid sequences were not affected. There was one base difference between obtained the BMAP-28 gene sequences and published corresponding sequences,which led to amino acid substitutions.2. After induction by IPTG, Rosetta bacteria containing pET28a-BMAP28 recombinant plasmid expressed fusion protein of the molecular weight of about 19.3kda, which was consistent with the expected size.3. Isolated pathogens from Mastitis in dairy cows which mainly concluded Escherichia coli, Staphylococcus aureus and Streptococcus, and purchased standard strain were used for antimicrobial testing of recombinant BMAP-28 expressed protein. The expressed protein had a significant inhibition to resistance E.coli, resistance Streptococcus, standard E.coli and standard S.aureus, but a weak inhibitory effect on resistance S.aureus.