| The extensive use of antibiotics has induced mutation of bacterial genetic material and selected d rug-resistant strains,resulting in the widespread spread of multidrug-resistant bacteria,which has laid a huge hidden danger for public health.Therefore,it is urgent to develop new antibacterial drugs.Antimicrobial peptide,as an endogenous polypeptide with immune function,plays an important role in biological development and immune process.At the same time,antimicrobial peptide is not easy to produce drug resistance,small molecular weight,broad-spectrum antibacterial and other advantages,making it one of the most potential alternative antibiotics.Insects live in various environments surrounded by pathogenic microorganisms and have a very strong innate immune system.Insect antimicrobial peptides are important members of innate immunity and play an important role in insect defense against pathogenic microorganisms.Coridius chinensis is a kind of edible and medicinal insect in Hemiptera.Previous studies have shown that C.chinensis has abundant antibacterial peptides in vivo,and the polypeptides and haemolymph isolated from C.chinensis have good antibacterial and anticancer cell activities.In this study,C.chinensis was selected as the material,and the defensin Cc Def2 gene was cloned based on C.chinensis transcriptome database.the expression pattern of Cc Def2 gene in C.chinensis,the structural characteristics and antibacterial activity of Cc Def2 mature peptide were studied,which laid a foundation for the physiological function research,development and utilization of Cc Def2.The results are as following: 1.Cloning and bioinformatics analysis of Cc Def2 geneBased on the data of C.chinensis transcriptome,the Cc Def2 gene was screened by relevant bioinformatics methods and defensin structural characteristics.the c DNA sequence was cloned and verified by RT-PCR technology.its ORF contains 351 nucleotides(nt),encoding 116 amino acids(aa),and the mature peptide segment has 43 aa,which has the molecular structural characteristics of insect defensin family.its molecular weight is 4.70 k Da,theoretical isoelectric point is 9.10,which is a hydrophilic cationic polypeptide with 5 net positive charges.It contains 6 conserved cysteine residues,forming 3 disulfide bonds,forming a high-level structure of CS??-motif with 1 ?-helix and 2 ?-folded sheets.Cluster analysis showed that Cc Def2 was closely related to Palomena prasina defensin,and their amino acid sequence similarity was as high as 74.42%.2.Analysis of expression pattern of Cc Def2 geneThe expression pattern of the Cc Def2 gene was detected by Real-time fluorescent real-time PCR(RT-q PCR).the results showed that Cc Def2 gene was expressed in all developmental stages of Coridius chinensis,and the expression level in 5th instar nymph was significantly higher than that in egg stage,1st-4th instar nymphs and adults.Analysis of the expression patterns in different tissues shows that the tissues with the highest expression of Cc Def2 gene is fat body,followed by haemolymph,and the expression levels in other tissues are relatively low.When the adult C.chinensis was induced by bacterial injection,the expression level of Cc Def2 gene reached the highest level when it was up-regulated to 12 hour,and then slowly down-regulated to the level before induction.3.Antibacterial activity of synthetic peptide of Cc Def2The antibacterial activity test results showed that the synthetic peptide of Cc Def2 had strong antibacterial activity against Staphylococcus aureus,and had no antibacterial activity against other gram bacteria tested.It could significantly inhibit the growth of S.aureus at a concentration of 40 ?g/m L,and could kill S.aureus at a concentration of 80 ?g/m L.Cc Def2 synthetic peptide has good tolerance to temperature and p H,and can still maintain the original antibacterial activity when the temperature treatment interval is-20-40? and the p H treatment interval is 3-10.It is sensitive to ionic strength,when Na Cl concentration is 10%,bacterial survival rate is 90.20%,Cc Def2 polypeptide loses most of its antibacterial activity against S.aureus.4.Construction and identification of recombinant vectorThe nucleotide sequence of the designed defensin Cc Def2 was optimized,and the GC content was reduced from 51.38% to 46.25% by replacing rare codons with synonymous codons according to the codon preference of Pichia pastoris.the Codon adaptation index,CAI)of the optimized gene was increased from 0.71 to 0.90,and the percentage of low frequency codons(< 30%)based on the host organism was reduced from 2% to 0%.After optimization,p MD18-T-Cc Def2 and p PICZ?A were double-enzymatically cleaved with Xho I and Xba I respectively,the target fragment with complementary sticky ends and the carrier skeleton were successively transformed into recombinant expression vector p PICZ?A-Cc Def2 by T4 DNA ligase,and the positive clones were selected and verified by colony PCR,double-enzymatically cleaving and sequencing to successfully construct recombinant expression vector p PICZ?A-Cc Def2.5.Expression of recombinant Pichia pastoris and determination of antibacterial activityIn order to realize the exogenous expression of Cc Def2,after linearizing and phosphorylating the recombinant plasmid with restriction enzyme Sac I,the linearized recombinant plasmid is electrically transformed into Pichia pastoris competent cell GS115,and the recombinant plasmid is integrated into Pichia pastoris genome.the positive recombinant strain GS115/p PICZ?A-Cc Def2 is screened through antibiotic screening,Mut+ phenotype screening,bacterial liquid PCR verification and genome PCR product sequencing verification.The positive recombinant strain was induced by 0.5% methanol.After the expression product was concentrated and detected by Tricine-SDS-PAGE,specific bands of about 5.5 k Da were found in the fermentation supernatants of 72 h and 96 h fermentation.The concentrated expression product has antibacterial activity to Gram-positive bacteria detected,and it has a the antibacterial activity against Staphylococcus aureus.However,it has the best antibacterial activity against Gram-negative bacteria. |
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