Cloning And Expression Of Antibacterial Peptide 40kDa Fetidin Gene Of Earthworm Eisenia Fetida

Antibacterial peptides are kind of small molecular peptides with antibacterial activity, coded by gene then translated by ribosome in natural immunity system of many species. Earthworm formed the mechanism for resisting external microorganism attack, produced many types antibacterial peptides in the passed several hundred million years evolution.They purified the two protiens, named fetidins, apparent molecular mass 40kDa and 45kDa respectively,to homogenity from dialysed coelomic fluid,and validated its antibacterial activity against Aeromonas hydrophila and Bacillus megaterium. Fetidins constitute the major coclomic fluid peptidic components (20% of total proteins) and are released from the chloragogue cell of the Eiseniafetida. It also can inhibit the growth of Staphytococcus aureus and Spiroplasma citri. The Fetidins' antibacterial characteristic has provided the basis as an external use antibacterial medicine and an antibiotic for agriculture. The aim of this reseach is to gain recombinant 40kD fetidin by gene cloning.We have extracted total RNA from earthworm Eisenia fetida, got cDNA fragment after RT-PCR. Then the DNA fragment is cloned in pMD18-T vector, and sequencing validate it include the full-length cDNA of 40kDa fetidin. We amplified the CDS of 40kDa fetidin by PCR, then digested it by restrict enzyme, jointed it with pKW32 and pMXB10 using T4 DNA ligase, to construct the recombinant pKW32-fetidin and pMXB10-fetidin. The confirmation is gained from two ways, including digestion by double enzyme and PCR identification. Transform pKW32-fetidin into DH5a and transform pMXB10-fetidin into ER2566, induce the recombinant to express products. The pKW32-fetidin is not stable in inheritance in DH5a. SDS-PAGE and metal affinity chromatography showed that fetidin was expressed. The recombinant proteins form inclusionbody in cytoplasm. Dissolve the inclusionbody with urea and refold it with metal affinity chromatography. The refold recombinant protein has certain antibacterial activity. We need to optimize the method for the refolding and the potential factor for determinating the antibacterial activity of recombinant protein.