| Magainin-Melittin Hybrid, an alkaline antimicrobial peptide, is produced by an active synthesized gene. Previous study done by Huang Yin(1999) reported that the hybrid shows antimicrobial activity. However, since the hybrid may kills its hosts cells,E. coli, in which it expresses, the hybrid gene produces little amount of peptides. In addition to having small molecular weight of about 2.9Kda, isolation and purification of the hybrid is difficult. This study is designed to cope with these issues. PCR is used for modifying the hybrid gene. On one hand, the gene will be improved by mutation. On the other hand, the improved gene inserts into the expression vector which contains an intein gene, fusion protein will thus be produced. It is beneficial to isolation and purification of the hybrid. Basing on the improved gene sequence, the forward and backward primers are designed. A mutation site and a Nde I identifying sequence are designed to be contained in the forward primer. The termination code is deleted from, and an ATG code and an EcoR I identifying sequence are the put into the reverse primer. The original gene acts as a template and PCR works for amplifying the improved gene. After being digested by Nde I and EcoR I 昦nd collected from alcohol precipitation method, PCR products are then cloned into the expression vector-pTYB~. Afterward, recombinant plasmids are transformed into E. coli hosts- ER2566. Expected sequence of the cloning gene is determinated by 3 using DNA sequencing analysis. Being induced by IPTG, plasmid expresses in host cells. Products are purified on IMPACT-T7 protein purification system and are detected by Tricine-SDS-PAGE. A specific bandof fusion protein is shown and molecular weight of 55Kda is determinated. This result demonstrates that the improved gene expresses in its host cells-ER2566. Moreover, purified peptides show antimicrobial activity when it is tested by using E.coli K12D31 as an indicator on plate diffusion method.
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