Expression Of Antimicrobial Peptide Tachyplesin Ⅰ From Tachypleus Tridentatus In E. Coli And Preparation Of Its Polyclonal Antibody

Antibiotic peptide are a group of small molecule peptides produced by plants, insects, amphibians and mammals including human beings. These peptides have many biological activities including antibacterial, antifungal, antivirus, antitumor and antiparasite. In order to avoid products on the toxicity of the engineering bacteria, guarantee the stability of expression and the right to effectively detect expression products, we do this research..The purpose of this research is to express the TachyplesinⅠfrom tachypleus tridentatus in E.coli and prepare its antibodies because of the very goodbiology activeness of The Chinese horseshoe crab antibacterial peptide.Expression of antibacterial peptide tachyplesinⅠfrom tachypleus tridentatus in E.coli. Cloning the tachyplesinⅠfrom the eukaryotic expression vector of The horseshoe crab antimicrobial peptide tachyplesin I. The PCR product was linked pMD18-T vector conversion, digestion and DNA sequencing. The DNAStar software showing that the tachyplesin I was consited 27 aa, the molecular weight was 2267.78Da, the charged number(ch) was + 5.78, the isoelectric point was (pI) 9.86.The carrying protein DHFR was digested from intermediate vector pQE-40 ,and then DHFR was linked to pQE-80L. Construction of antimicrobial peptide tachyplesin I from the horseshoe crab prokaryotic expression vector and then obtained the prokaryotic expression vector pQE-80L-DHFR-TPⅠ. Positive plasmid was identified by double restriction enzyme digestion and PCR, and then transformed into thecompetent cell and induced with IPTG.The results showed that the tachyplesinⅠwas expressed in E.coli. The DNAStar software showing that the recombinant protein DHFR-TPI was 25968.17Da, the charged number(ch) was + 12.11, the isoelectric point was (pI) 9.48. Examination of the SDS-PAGE, the recombinant protein DHFR-TPI mainly existed in form of inclusion bodies.The proteins are 2.945 mg/mL.Purification of recombinant proteins and detection of its antibacterial activity . The optimal conditions were determined that the induction time was 4～5 hours, the temperature was 30℃, the pH was 7.4 and the IPTG concentration was 1.5 mmol/L,under this conditions the expressed recombinant protein,the target protein was about 52.97% in the total bacterial protein with TLC . Best induced conditions for the use of a large number of engineering bacteria cultivation , on the basis of the SDS-PAGE and purified protein with bag filter and electroeluting. BandScan5.0 software showed that purified protein DHFR-TP I was about 2.5mg /mL,the purity was 91.5%; the purity of DHFR was 75.1%. Rehabilitation of the purified protein,and antibacterial experimental showed that it could inhibit Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans and other antimicrobial, antibacterial circle was 21,13,15,12mm..Preparation and identification polyclonal antibodies of recombinant peptide . The Rex adult was immuned by purified recombinant protein DHFR-TPI, DHFR, PBS control.First,complete Freund's adjuvant wasused, multi-point injection subcutaneously on the back, 2.5mg/mL; Second, thrid and the strengthen time , Incomplete Freund's adjuvant was used ,back muscles and armpit injection. An indirect ELISA was developed.The results showed that the optimal coating concentration was 2.4μg/mL , the optimal coating time was 12 h at 4℃subsequent to 2 h at 37℃,and the optimal concentration of Second anti-HRP was 1:40 000. The dilution of antibody against DHFR-TPI after strengthen injection was more than 1:250 000 .The result shows thatantisera DHFR-TP I was difference with DHFR antisera by SPSS.The results showed rabbit anti-serum DHFR-TP I can work well with the expression of specific proteins DHFR-TP I integration by western-blotting. Fusion protein DHFR-TP I has a strong reaction in rabbit.In this study, the antimicrobial peptide tachyplesin I from Chinese horseshoe crab was cloned , expressed in E.coli and prepared the polyclonal antibodies by the purified recombinant peptide.All these made good foundation for research of affinity chromatography ,cecropin detection, location and mechanism of the antimicrobial peptide in the future.