Fusion Expression Of Antimicrobial Peptide Thanatin And Baculoviral Polyhedron And It’s Eukaryotic Expression

Thanatin, containing21amino acid residues, is a linear antimicrobial peptide which wasfirst discovered in insects Podisus maculiventris, and has high sequence homology to frogskin antimicrobial peptides of the brevinin family. The insect and frog peptides have incommon a C-terminally located disulfide bridge delineating a cationic loop. The cationicpeptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrumobserved so far for an insect defense peptide. It is difficult to obtain large amount ofpeptides from the nature organization, and it’s high costly to abtain peptides from chemicalsynthesis method. Genetic engineering methods of production of antimicrobial peptideshas become a research hotspot in recent years.This is the first time to build a fusion prokaryotic vector which combined with Bombyxmori nuclear polyhedrosis virus Polh gene and Thanatin gene. The fusion protein wasexpressed in E. coli as inclusion body. After preliminary purification (cutting withhydroxylamine hydrochloride), it is found that the peptides has antibacterial activityagainst DH5a. This is the first time to express Thanatin gene in Baculvirus express system.We have obtained the following results.1. We optimized the thanatin gene as codon preference in Eoli. We obtain Thanatin andpolyhedron fusion gene using overlap PCR methods. The fusion gene was cloned into theexpression vector pET28a (+) to construct the expression vector pET-28a-Polyhedrin-Thanatin. The plasmid were transformed into E. coli BL21(DE3) and induced by IPTG.The expression product was analyzied by SDS-PAGE electrophoresis and Western blot.The results which was consistent with the theoretical value (35kD) confirm the expressionof Polyhedrin-Thanatin fusion protein. The target fusion protein was expressed as inclusionbody. The expression level was accounting for30%of the total bacterial proteins in6hafter induced by IPTG. The purified fusion protein was cut by hydroxylaminehydrochloride. The purified Thanatin was showed antibacterial (DH5a) after antibacterialtest.2. We expressed Thanatin gene in BmN cell by Bac to Bac express system. First, theThanatin gene was cloned into the baculovirus transfer vector pFastBacI. The recombinantplasmid pFastBacI-Thanatin was transformed into E. coli DH10Bac competent cells. After recombinant in DH10Bac, the recombinant Bacmid was extracted and infected Bombyxmori cells (BmN4) with Lipofectin-mediated. The recombinant baculovirus was obtainedand futher infected Bm cells to express the target protein. The cell lysate was obtained forantibacterial test. The results showed that the cell lysate can inhibit the growth of E.coliDH5a, which provide a basis for further separation Thanatin and other experimental study.