CDNA Cloning And Construction Of Recombinant Expression Vectors For Fish Antibacterial Peptide BPI And LEAP2

The abuse of antibiotics has led to increased evolution of drug-resistantpathogenic strains, and it is necessary to find new drugs with structural uniqueness tofight them. Antimicrobial peptides generally are some small molecule peptidesproduced by the cells of living organisms. They play an important role in the host’simmune response against a wide variety of microbial invasion, such as bacterial, fungal,viral, and other pathogenic infections and they are considered to be good substitute fortraditional antibiotics. Fish are a major component of the aquatic fauna. Aquatic sourcescontain thousands of fish species which can secret AMPs with structural differences,among which two kinds of fish antimicrobial peptides were studied in this study,bactericidal/permeability increasing protein（BPI）from channel catfish and commoncarp and liver-expressed antimicrobial peptide2(LEAP2) from channel catfish. Thereare a few reports both home and abroad on the recombinant expression of these twoantimicrobial peptides from fish.BPI, an antimicrobial protein expressed in organism cells, shows particularantimicrobial activity against gram-negative bacteria. BPI contains two domains,N-terminal domain and C-terminal domain. The antibiotic and endotoxin-neutralizingfunctions of BPI are attributed to the N-terminal domain. The present study wasperformed with reference to the report for the recombinant DNA expression of humanBPI. The cDNA fragments “BPINtd” and “BPINtdcf” encoding the N-terminal activedomain of BPI were cloned from the gill of channel catfish（Ictalurus punctatus）byRT-PCR and nested PCR.“BPINtdcf” had a terminator codon “TAA” at the end of thefragment. These fragments encode a peptide consisting of175amino acid residues, inwhich63residues can form a three-dimensional apolar lipid-binding pocket which isused to bind lipopolysaccharide（LPS）located in outer membrane of gram-negativebacteria. The comparison of the channel catfish BPI with the BPIs from other organismsshowed that40conserved amino acid residues appear at the N-terminal domain, andamong them9highly conserved residues located in the LPS-binding region. ThepET-32a（+）and pET-28a（+）with different characteristics were chosen as theprokaryotic expression vectors to construct the prokaryotic expression system.“BPINtd”fragment was inserted into these two plasmids respectively. The colony PCR, EcoR I and Hind III restriction endonuclease digestion and DNA sequencing demonstrated thatthe“pET-32a-BPINtd” and“pET-28a-BPINtd” recombinant expression plasmids wereconstructed successfully. Meanwhile,“BPINtdcf” fragment was inserted into pET-28（a+）plasmid. The colony PCR, Nde I and Bam HI restriction endonuclease digestion andDNA sequencing demonstrated that “pET-28a-BPINtdcf” recombinant expressionplasmids were constructed successfully.Further, a cDNA fragment“BPINtdcp” encoding the N-terminal active domain ofBPI was cloned from the gill of common carp by RT-PCR and nested PCR. Thisfragment encodes a peptide consisting of211amino acid residues, in which63residuescan form a LPS binding domain.“BPINtdcp” fragment was inserted into pET-28a（+）plasmid. The colony PCR, Nde I and Hind III restriction endonuclease digestion andDNA sequencing demonstrated that “pET-28a-BPINtdcp” recombinant expressionplasmids were constructed successfully.Liver-expressed antimicrobial peptide2(LEAP2) was first obtained fromultrafiltration products of human blood, which had two disulfide bridges and wasexpressed by liver. A LEAP2gene which encoded a41-amino-acid mature peptide wasamplified from the liver of channel catfish (Ictalurus punctatus) by RT-PCR. It wasnoted that when the channel catfish mLEAP2was compared with those frompoikilothermal fish and homoiotherms, fourteen conserved residues occured, amongwhich four highly conserved cysteins residues occurred at C-terminal regions of thesemLEAP2s and formed two disulfide bridges, suggesting that these four residuespossibly related to antimicrobial activity of LEAP2. Subsequently, the recombinantexpression plasmid “pET32a-mLEAP2” was constructed and the mLEAP2gene wasfused with a trxA partner with a6x His-tag and an enterokinase site. The fusion protein“trxA-LEAP2” was successfully expressed at25°C in E. coli BL21(DE3) by adding0.7mmol/L IPTG after a16-h induction. Tricine-SDS-PAGE showed that the fusionprotein existed in both supernatant and inclusion body after sonication andcentrifugation. Highly purified fusion protein was successfully obtained by ImmobilizedMetal Affinity Chromatography (IMAC).Further, a new fragment “mLEAP2kex2” with Xho I and Xba I restriction sites wasobtained with “pET32a-mLEAP2” as template. A Kex2cleavage site “AAAAGA” wasincluded in the “mLEAP2kex2”, which can cleavage the α-factor signal sequence fromthe fusion protein. Thus, mLEAP2can be expressed with a native N-terminus which isof great importance to its antimicrobial activity.“mLEAP2kex2” fragment was theninserted into pGAPZαA plasmid. The colony PCR, and DNA sequencing demonstratedthat pGAPZ-mLEAP2kex2”recombinant expression plasmids were constructed successfully, which laid foundation for its expression in Pichia pastoris GS115.This paper can help people get a better understanding of AMPs from fish andsearch for novel AMP drugs to treat drug-resistant pathogens which can be used by thepharmaceutical industry, breeding industry and agriculture.