Antimicrobial Activity Analysis And Gene Expression In E.coli Of 29aa-peptide From Earthworm

Antimicrobial peptides are a kind of small molecular peptides with antimicrobial activties, which play an important role in the innate immunity system of many species. Earthworm living in foul environment developed unique mechanism of resisting external microorganism attack and produced many types antimicrobial peptides in biological evolution for a long time..A peptide corresponding to 29 amino acids of LumbricinⅠ(6-34) having high similarity to amino acid sequence of EAP-1 was synthesized by chemical method.The colony forming unit technique was used to study antimicrobial spectrum, thermostability, action time, and minimum inhibitory concentration(MIC) of the 29aa-peptide. The result showed that the 29aa-peptide has the following characteristics:1)It has a wide antimicrobial spectrum, which can inhibit not only common bacterias such as Escherichia coli, Staphylococcus areus, but also fungi such as Saccharomyces cerevisiae, Myrothecium roridum,etc; 2) Its antimicrobial activity was so powerful that a concentration of 0.009 mg/mL could significantly suppress the growth of Escherichia coli;3) It has good stability, so its antimicrobial activity maintains at pH down to 2.2 or up to 11 and keeps 80% of activity after boiling for 10 min.According to the codon usage of E.coli, a single strand DNA encoding 29aa-peptide was synthesized.The gene coding for 29aa-peptide was obtained by two step PCR and was cloned into the pMD19-T vector, the sequence cloned was identified.A fusion expression vector pGEX-4T-2-29aa was constructed and was transformed to E.coli DH5a. After induction by IPTG the expressed product GST-29aa was digested by thrombin. However antimicrobial activity of the free 29aa after excision GST lebel was not detected.At the same time, a non-fusion expression vector pBV221-29aa was also constructed and was transformed to E.coli DH5a and DE3.After induction at 42℃the non-fusion expressed product 29aa was determined by Tricine-SDS-PAGE, and it showed that the yield of 29aa is very low, but it has antimicrobiol activty.