Manufacture And Application Of Six Cecropins Antibacterial Peptides From Gene Engineering

Abuse of antibiotics not only cause serious drug resistance of some pathogenic bacterium but also has bad impact on public hygiene such as drug remnant etc. Antibacterial peptide rears the obvious advantage in new medicine research because it composes the broad-spectrum antibacterial activity efficiently and special antibacterial mechanism is studied widely. With the resent researches, the ABP is a broad-spectrum antibiotic and can inhibit and kill resistant bacteria strain. Consequently, it is the most promising substitute for traditional antibiotics. Antibacterial peptides (ABP) include a series of small peptides with positive ions most of which have abroad-spectrum antibiotic and they are significant ingredients during the course of congenital immunity in most creatures. The purpose of this research is to develop recombinant ABP through the technique of gene engineering and protein engineering with the property right reserved and study the activity in vitro and in vivo. The study was unfolded through the following aspects.1. Cecropin B gene was achieved through gene splicing by overlap extension (SOE). Especially a Kex2 signal cleavage site was fused in 5' end of the antibacterial peptide gene. Cecropin B gene was subcloned and ligated tandem. The modified cecropin B and it's tandem genes cloned into pPICZa -A vector to construct the recombinant expression vectors. The recombinant expression vectors were linearized by SacI and transformed into Pichia pastoris SMD1168 strain by electroporation. Positive clones were screened and those clones were fermented in flask. Tricine- SDS- PAGE showed that the Cecropin B protein could be secreted into culture leading by a - factor came from pPICZ a- A. 2. According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mag gene was synthesized and cloned into pPICZa-A to construct the recombinant expression vector pPICZa-A-CA. The pPICZa-A-CA was transformed into yeast host strain SMD1168. Under the control of the promoter AOX' (alcoholoxidase'), an approximately 1.9 kD cecA- mag protein was expressed. The hybrid antibacterial peptide had a broad -spectrum antibacterial activity on both gram positive and negative bacteria, especially showed potent antibacterial activity against ampicillin resistant bacteria, such as pathogenic E.coli. In addition, based on these characteristics, the recombinant antibacterial peptide cecA-mag was mutanted by the recombinant PCR. In this way, the three mutants were gained. According to agarose diffusion assay, antibacterial activitives of cecA-mag-mu-1 and cecA-mag-mu-2 are 1.2 times than cecA-mag, cecA- mag -mu-3 is 1.5 times than cecA- mag.3. The requirements for the flask- shaking culture fermentation of the ABP's recombinant pichia pastoris were optimized. It was found the expressed level of the recombinant antibacterial peptide, could be gained when the density of the seed yeast amounted to OD600 value 5-6, when pH value of the culturing BMMY medium was 6.0, when yeast was cultured under 28℃for 60 hours, 1.0% methanol was supplemented into the medium. According to agarose diffusion assay, the recombinant geneic ABPs had a broad -spectrum antibacterial activity on both gram-positive and gram-negative, especially showed poptent antibacterial activity against ampicillin-resistant bacteria. In addition the heat-stable and acid-stable characteristics of ABPs peptide were examined. We evaluated their's antibacterial effect, using the growth curve of bacteria after bacteria being treated with different concentration of cecropinB and cecA- mag.4. Based on these characteristics, the recombinant antibacterial peptide display application foreground in the field of prevention of disease, additives of animal feedstuff and so on. Using one-day chick, the effective of treating bacterial infection were studied. MLD₁₀₀ of a strain of Escherichia coli to chick was determined preliminary. In the treating experiment, the chick treated with different concentration recombinant antibacterial peptide cecropinB injection and oral administration were treated after 24 h of infection with the dosage of MLD₁₀₀ of Escherichia coli. This treatment was lasted five days. The results showed that the recombinant antibacterial peptide cecropinB has preferable bioactivity in vivo. Especially, the result of oral treatment with high dosage cecropinB is optimization. The death rate of treatment with antibacterial peptide cecropinB and Ciprofloxacin degraded obviously.5. Insects produce a variety of antibacterial peptide that act synergistically to kill invading microoganisms. Antibacterial peptide was also found in other animals. To study the effect of antibacterial peptide cecropinB on the growth performance in chickens, two hundred fifty 1-day-old chickens with similar weight body were separated into experimental and control groups at random. 2 mL,4 mL and 6 mL of antibacterial peptide (ABP) cecropinB were administered hydroposia into each experimental group chicken every day, the 2 g Ciprofloxacin control group' s chicken at the same age, respectively. Five groups of chickens were fed under the same conditions for 7 weeks. The index of thymus, Fabricius and spleen were determined at age of 7,14,21,28,35,42and 49 days respectively. The average daily weight gain of the experimental group chickens was significantly higher than the control group chickens. The index of thymus, Fabricius and spleen of the experimental group chickens were higher than those of the control group chickens. So, antibacterial peptides cecropinB could enhance the growth performance and improve immune function of chickens.6. Effect of geneic engineering antibacterial peptide cecropinB on intestinal microbial community in chick was studied by traditional method and denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA. One-day-old chickens were allocated randomly into five groups, fed water which interfuse three different concentration cecropinB (treat group) or antibiotic (medicine group) and the negative group has no additive separately. DGGE analysis showed there were no abnormity of the microflora in the colon. In conclusion, cecropinB had no observed adverse effect on the intestinal microbial community in chickens.