Modification,Cloning And Prokaryotic Expression Of The Gene Encoding Antibacterial Peptide PolyphemusinⅡ From Tachypleus Tridentatus

Tachyplesin , an antibacterial peptide in the blood of horseshoe crab, has broad-spectrum antibacterial ability and inhibitive effect on tumor cell and virus and received the broad attention of scientist . Due to poor quantity of the horseshoe crab and the difficulty in breeding of horseshoe crab , the tachyplesin isolated from horseshoe crab can not meet the supply ,so it is essential to use molecular biology technology to develop an engineering bacterial to produce tachypiesin effectively . To get the objective product, the whole blood was extracted from the heart of the horseshoe crab firstly and total mRNA isolated from it with Trizol .The primers were designed based on the reported sequence pf antibacterial peptide polyphemusinII from America horsecrab .The modification of the antibacterial peptide gene , including the C-terminal amide hydroxylation, was adapted to promote its ability and stabilization , followed by RT-PCR amplification . The amplified gene was be cloned into pMD18-T, and named as pMD18-T-rr. Then, the sequence was determined. Secondly , the plasmid pMD18-T-rr was digested with Ndeâ… and EcoRâ… ,and then the target fragmemt was linked into prokaryotic expression vector pET28c(+). The recombinant plasmid was transformed into E.coli JM109 strain. After identification, the recombiant plasmid was transformed into host strain E.coli DE3, induced by IPTG for expression. An expressed protein with the size of 2.5KD, was identified by SDS-PAGE and it accounted for 27.21% the total protein of the germ. Besides, the expressed protein was reactive with the positive serum of nature tachyplesin. Moreover, The expressed product showed obvious antibacterial ability as determinded by MIC essay. In conclution , polyphemusinâ…¡gene was modified successfully in this study. And when it was transformed into E.coli, the gene was effectively expressed. The expressed product showed strong antibacterial activity. Hence ,this study lay the basis for large scale production and application of the antibacterial peptide polyphemusinâ…¡.