Synthesis And Secretory Expression Of The Origined Human And Hybrid Antimicrobial Peptides From Lactococcuslactis

BackgroundAt present, with the wide application of antibiotics, bacterial resistance to antibiotics and drug-resistant spectrum is rapid increase and expanding. The antimicrobial agents based on the role of antibiotic and existing microbial species will face unprecedented crisis. In addition, intestinal bacterial flora balance has closed relationship with human health, dysbacteriosis could induce a variety of diseases on host, especially intestinal diseases, such as diarrhea, IBD. Antibacterial peptides, also called. Antimicrobial peptides, are kinds of broad-spectrum resistance activities peptides to bacteria, fungi, viruses, protozoa, and tumor cells by a variety of biological cells specific genes encoding and outside conditions induced. AMPs, as important components of natural immune systems, could be a new effective way to control infection because of their broad spectrum antibacterial activities and the characteristic of drug-resistant difficult creation. Now there are more than 1500 kinds of peptides which have collected into the database and confirmed, source types include such as bacteria, plants, insects, amphibians, invertebrates and mammals. The humanity can also product the AMPs by itself. There are two kinds of human antimicrobial peptide families which have more important researches and the extensive effect. They are cathelicidins family and defensins family.So far, LL-37 was found the only kind of antibacterial peptide of Cathelicidins family in the human body. It widely exists in the neutrophils of epithelial cells secretion of the gastrointestinal tract, urinary tract, respiratory tract, etc. The secretion of LL-37 has increased obviously when tissues are inflammation, infection, and damage. LL-37 has broad-spectrum antimicrobial activity for a variety of gram-positive bacteria, negative bacteria and fungi. At the same time, as immunomodulatory factor, LL-37 can release IL-8 factor by keratin cells to influence the host inflammatory response. Alpha human defense 5 (HD5) is an important antimicrobial peptide in the defenses family. It mainly expressed in Paneth cells of the small intestine, and it can increase secretion expression with factors stimulating by inflammation, infection and malignant cell transformation in the intestinal tract. HD5 not only has efficient broad-spectrum antimicrobial activity and adjust the immune protection, can also be antiviral like as HIV and HSV. In view of a variety of active function of antibacterial peptides, they get more attentions in the aspect of developing new antibacterial drugs and regulating immune.Nowadays, AMPs extracted mainly from natural resources, they were restricted wide application by the high cost, low yield and process complicated. And the method of chemical synthesis was relative expensive to apply to clinical difficultly. AMPs using genetic engineering technology production is of great significance. Lactic acid bacteria, as probiotics which closely related to human life, was recognized as generally regarded as safe(GRAS). It not only can inhibit the growth of harmful bacteria, also can promote the synthesis of nutrients, improve the gastrointestinal function, and treat the gastrointestinal diseases. This study selects the nisin-controlled gene expression(NICE) system of Lactccoccus lactis (L. lactis).It is the most widely used and strong controlled in the expression system of Lactobacillus. We artificially synthesized objective antibacterial peptide gene to obtain the secretion expression protein of origined human and hybrid antimicrobial peptides by the Lactobacillus expression system and the method of gene splicing by overlap extension (SOE). And we studied the antibacterial activities in vitro to realize the synergy of probiotics and antibacterial peptides, and provided the basis for subsequent animal and clinical experiments.Objective1. This study designed the hybrid antimicrobial peptides with better activity called LLD1 and LLD2 according to the gene sequences of the natural and human AMPs LL-37 and HD5.2. The four AMPs, called LL-37, HD5, LLD1 and LLD2, were synthesize in vitro. And the restructuring secretory expression vector pNZ8148-sp-LL37/HD5/LLD1/LLD2 would be built, eventually they secreted proteins in L. lactis NZ9000.Methods1. The selection of antimicrobial peptides with LL-37 and HD5 as a blueprint of hybrid design, their gene sequences can be found in GenBank. To optimize their gene sequence according to the preference codon of the L. lactis, so that they can be better expressed in L. lactis. Finally, this study design the two gene sequences of hybrid antimicrobial peptides LLD1 and LLD2, and a total of four AMPs are analysis and prediction through bioinformatics methods in secondary structure, amphiphilic, isoelectric point and transmembrane region, to compare their antibacterial activity.2. According to the L. lactis expressed preference codon database, we obtain the four antibacterial peptides optimized gene sequences. Each AMPs gene sequence is respectively divided into four segment primers with 18-20bp overlapping. Using overlapping extension PCR synthesizes antimicrobial peptide gene sequences, and insect the restriction enzyme sites Kpn I and Xba I in the gene sequences. Finally the four synthesis AMPs genes ligate respectively the pMD19-T simple plasmid to construct recombinant plasmid, and transform into Escherichia coli (E. coli) Top 10 by chemical conversion. After getting positive clonings, extraction of plasmids, they are validated by restriction enzyme digestion and gene sequencing.3. The four kinds of restructuring AMPs of verified correctly are double digested with restriction enzymes. The peptide sequences with gel extraction ligate the secretory expression vector pNZ8148-sp plasmid, and then electroporation into L. lactis NZ9000, building four kinds of recombinant Lactobacillus. Through bacterium solution PCR method, restriction enzymes digestion method and gene sequencing to identify positive transformation. Pick the recombinant Lactobacillus of verified correctly to culture, after nisin inducing, four kinds of antibacterial peptides secreted expression respectively. The expression products are identification with Tricine-SDS-PAGE electrophoresis, Western Blot and Dot Blot, ultrafiltration and concentration by using the ultrafiltration centrifugal tube which is intercept molecular weight for 30KD and 1KD in stages. The purified supernatant fluid can be experiment of bacteriostatic activity.Results1. Through the preference codon of L. lactis, we redesign antimicrobial peptides LL-37 and HD5 gene sequences, and as female parent, design two new hybrid antibacterial peptides LLD1 and LLD2. Through bioinformatics analysis, the antibacterial peptides LLD1, LLD2, HD5 and LL-37 all belong to cationic alpha helix structure type. Their amino acid residues have amphiphilic at C terminal. The antibacterial peptide LLD2 has obvious amphiphilic, and the strongest hydrophobic at its C side. Four peptides have no obvious transmembrane helical regions, it means that they may not be traditional transmembrane protein, their mechanism of action may be related to the tramsmembrane holes by combined with cell membrane by electrostatic forces. By a series of analysis, for the entire, the heterozygous peptide LLD2 may has stronger antibacterial activity than other three of antimicrobial peptides.2. Using the design synthesis complementary primers to success obtain the AMPs full-length gene sequences by overlap extension PCR. This experimental synthesizes four AMPs LLD1, LLD2, HD5 and LL37, their gene length is respective 100bp,112bp,115bp and 130bp. The electrophoresis results are consistent with the expected amplified fragment size. After restriction enzyme digestion identification and gene sequencing, the four AMPs sequence are shown to verify the correct. That has been successfully cloned LLD1, LLD2, HD5 and LL37 the four fragments, the recombinant plasmids named respectively pMD19Ts-LLD1, pMD19Ts-LLD2, pMD19Ts-HD5 and pMD19Ts-LL37.3. Testing by bacterium solution PCR, the expected synthetic fragment size of the four carriers pNZ8148-sp-LLD1/LLD2/HD5/LL37 is respective 400bp,412bp, 415bp and 430bp, as same size with a single bright stripe in the corresponding lane position. It means that recombinant Lactobacillus transform into success. According to the results of the restriction enzymes digestion and gene sequencing identification, it showed that the recombinant Lactobacillus which can secrete antimicrobial peptide protein have been built successfully. Eventually, we obtain four recombinant L. lactis, respectively named NZ9000-SLLD1, NZ9000-SLLD2, NZ9000-SHD5 and NZ9000-SLL37. Their fermentation supernatants identify by the Tricine-SDS-PAGE gel electrophoresis, and all can be see the bright stripe at between 3.5-5KDa positions, consistent with the expected antibacterial peptide protein sizes. And by the inmmune detection, it is confirmed that the four kinds of antibacterial peptides protein are correct secretory expression. The supernatant expression products with purification and concentration are tested their activities in the bacteriostatic experiment. We can see four kinds of antibacterial peptides are all showed bacteriostatic effect. But in the process of the bacteriostatic experiment, we found that the bacteriostatic effect are unstable, bacteriostatic repeatability is not good. We consider that the antibacterial peptide proteins purity is not enough, and correlate with host protease degradation in cells. We plan to further strengthen the control of purification and extraction of the antimicrobial peptides, to ensure the stability of the samples.Conclusion1. In order to get a stronger antibacterial activity of antibacterial peptides, and to easily get the AMPs which for better treatment of diseases in human intestine, there is a series of bioinformatics research and hybrid design which is based on how to improve antibacterial activity. Molecular cloning technology is used to construct restructuring probiotics to express the purpose peptides continuously.2. This study designs the hybrid antimicrobial peptides LLD1 and LLD2 based on antibacterial peptide HD5 and LL37. Using bioinformatics software to analyse and predict in secondary structure, amphiphilic, isoelectric point and transmembrane region, we found that the isoelectric point of antibacterial peptide LLD1, LLD2, HD5 and LL37 is all greater than 7, their structure are based on alpha helix structure. And, the two hybrid peptides, especially LLD2, have better antibacterial activity than the mother peptides HD5 and LL37.3. This study synthesizes successfully four antibacterial peptide genes LLD1, LLD2, HD5 and LL37 by overlap extension PCR, and cloning in the pMD19-T simple carrier, obtains correct antibacterial peptide gene sequences.4. The study constructs successfully the recombinant secreted expression vectors pNZ8148-sp-LLD1/LLD2/HD5/LL37, and transforms into the probiotic L. lactis NZ9000. Eventually we build four kinds of recombinant genetic engineering bacteria NZ9000-SLLD1, NZ9000-SLLD2, NZ9000-SHD5 and NZ9000-SLL37, and induce proteins expression successfully. The supernatant expression products with purification and concentration are tested their activities in the bacteriostatic experiment. The result shows that all of them have bacteriostatic effect. And according the independent bacteriostatic experiment with different volume of the fermentation supernatant, we found that E. coli was basic inhibition growth when the supernatant reached 10mL. But due to lack of sample purification process, the purity of antibacterial peptides is not good enough, and their antibacterial effect is not very stable.5. On the basis of the existing research, there are three aspects for further study and improvement:(1) To improve the expression and stability of the antimicrobial peptides. (2) To do animal experiments which can verify the regulation on intestinal flora, and the defense effects of intestinal immune barrier. (3) To improve the design of the antibacterial peptide gene, which the antimicrobial peptides of more antibacterial active can be manufactured.