Studies On Preparation Of Antimicrobial Peptide From Horseshoe Crab (Tachypleus Tridentatus) With Gene Engineering Method And Its Biological Activity

Abstract: In order to preparation the antimicrobial peptide of Horseshoe crab(Tachypleus tridentatus)possessing several biological activities, which is nonpoison,no harmful remainder and not easy to produce drug resistance to bacteria, geneengineered germ of pronucleus and eukaryon were constructed, avoiding to destructthe ecological natural resources.Amebocytes were obtained from an adult male of Horseshoe crab(T. tridentatus).Immediately after centrifugation, the hemocytes were placed and milled in liquidTrizal. Total RNAs were prepared from hemocytes, and poly(A)+ RNAs werepurified using PolyATract mRNA purification kit with oligo ( dT ) -cellulosechromatography(Promega Corporation). The first strand cDNA synthesis was primedwith a hybrid oligo(dT)linker-primer and random primers and was transcribed usingmoloney murine leukemia virus reverse transcriptase (TaKaRa Biotechnology, Inc.).The synthesized cDNA was used as a template in subsequent PCR. The primers weresynthesized according to the Nucleotide base sequence of tachyplesins and Aminoacid sequence of polyphemusins from Limulus polyphemus, for want of Nucleotidebase sequence reported. The codons recognized preferentially by E. coli were used.PCR was performed in a Biometra personal cycler. Amplified DNA fragments wereligated into pMD18-T vector, and transformed into E. coli strain JM109 by thecalcium chloride method. The sequence was congfirmed by DNA sequencing.Redesign the primer to get the genes of KRN(source gene tachyplesins)and rr(source gene polyphemusins), avoiding TAA to code for Trp, which the C terminuswere replaced by AAC(codon of Asn) and the restriction site of NdeⅠwere addedto construct the recombinant expression plasmids pET-KRN and pET-rr with themethods as described previously and transformed into DE3. The DE3-pET-KRN andDE3-pET-rr were induced by IPTG to express antimicrobial peptides (KRN and rr).The expressing soluble products identifying with Tris-Tricine SDS-PAGE, is2650.22Da and 2912.41Da respectively, and existed in the supernatants ofrecombinant bacteria in breaking buffer. Thin layer scan analysis of the protein bandsindicated that the interest protein accounting for 8.6 and 6.1 in the total protein.In vitro susceptibility tests for the supernatants of indicated recombinant bacteriais performed to the Escherichia coli, Staphylococcus aureus, Salmonella typhi,Bacillus subtilis with mulitipe resistance. With the method of Kirby-Baller, the resultof KRN is 25, 22, 16, 25mm respectively and that of rr is 20, 21, 10, 18mmrespectively. There is no effect to Pseudomonas pyocyanea. With MicrodilutionMethod, the result of KRN is 3.57, 7.14, 3.57, 14.28μg/mL respectively and that of rris 3.70, 7.60, 3.70, 15.20μg/mL respectively.Although the antimicrobial activity is better, bacterial content is lower for toxicresponse. So that two yeast expression plasmid pPIC-KRY and pPIC-rrY containingtachyplesins and polyphemusins genes were constructed using the expression plasmidspPIC9 as vector. After the linearized recombinant plasmids had beenelectrotransformed into the competent yeast strain GS115 of Pichia pastoris...