Prokaryotic Expression Of Cecropin P1 And Antibacterial Activity Analysis

Issues such as drug residues and drug resistance have become increasinglyserious with the use of a large number of traditional antibiotics, and searching for novel antibiotic substitutes is extremely urgent. Because of their advantagessuch as the unique antibacterial mechanism and little drug resistance, antibacterial peptides have broad application prospects in terms of alternatives to traditional antibiotics and reducing drug resistance. Cecropin. an antimicrobial peptide.was firstly identified in Hyalophora Cecropia by Huhmark et al. and so far there are more than 30 kinds of cecropins reported, in which Cecropin P1(CP1)was firstly isolated from pig intestine by Lee et al. CP1 consists of 31 amino acids with a molecular weight of 3339 Da and has a broad-spectrum antimicrobial activity. But the natural yield of CP1 is limited and its separation and purification are difficult at a high cost, which greatly limits its clinical application.Therefore the production of CP1 through genetic engineering is of important significance.This study was designed to obtain antimicrobial peptide CP1 by using the prokaryotic expression system and to provide the theoretical and practical basis for the construction of genetically engineered bacteria strains and the large-scale production of CP1. Based on the amino acid sequence of the mature peptide of CP1, using codon preference of Escherichia coli. CP1 gene was designed andsynthesized by overlap PCR technique, and the recombinant plasmid pMD19-T-CP1 was constructed. The prokaryotic expression vector pCold TF and the recombinant cloning plasmid pMD19-T-CP1 were double-digested by restrictive endonuclease EcoR Ⅰ and Xba Ⅰ, respectively, and the recombinant expression vector pCold TF-CP1 was constructed using T4 DNA ligase. Then the pCold TF-CP1 was transformed into BL21 (DE3) competent cells. The positive transformants (recombinant engineering bacteria) were induced to express the fusion protein (rCP1) by IPTG. The tCP1 protein was purified by Ni-NTA column and desalted and concentrated by ultrafiltration. and then CP1 was released by enterokinase cleavage. The inhibitory activity of CP1 was finally detected by the thin agarose cavity diffusion method. The main results are as follows:1. The target CP1 gene was successfully synthesized by overlap PCR technique, and the recombinant plasmid pMD19-T-CP1 was constructed.2. The recombinant expression plasmid pCold TF-CP1 was successfully constructed and transformed into BL21 (DE3) competent cells to obtain the engineering strain of the recombinant plasmid.3. The engineering strain of the recombinant plasmid was induced by IPTG toexpress a soluble fusion protein, which accounted for more than 36% of the total soluble protein; the fusion protein was purified by Ni-NTA column with a purity of about 80%; the concentration of the concentrated protein after ultrafiltration was determined to be 3.5 mg/mL by means of ultra-trace spectrophotometric meter (NANODROP 2000); based on calculation, approximately 55 mg fusion protein per liter of bacterial culture could be obtained.4. It was confirmed by comparing with standard product that the CP1 was released from the fusion protein (rCPl) by enterokinase cleavage with a concentraton of (0.14 ±0.04 mg/ml). The results of antibacterial tests showed that CP1 had good antibacterial activities against Klebsiella pneumoniae. Escherichia coli. and Salmonella, but no antibacterial activity against Staphylococcus aureus was found.In this study, the fusion expression of antimicrobial peptide is achieved by a prokaryotic expression system and bioactive CP1 is released after enterokinase cleavage of the fusion protein, providing the theory and practice basis for the constructions of genetic engineering bacteria strains and large-scale productions of CP1 and other antimicrobial peptides.