| Antimicrobial peptides are a class of small peptides with biological activity, it can be induced in vivo. They are widely used in food and medicine, also identified as a class of environmentally friendly feed additives. Cecropin B is a thermally stable soluble polypeptide which was first isolated from the Super Warm body. It only includes 35 amino acids. Cecropin B has the stronger antimicrobial activity compared with many antimicrobial peptides isolated from nature. It’s harmful to bacteria, fungi, viruses, even kills them.Methionine is usually the first limiting amino acid, specially those containing high levels of plant protein sources. Thus adding methionine in animal diets can improve the utilization of plant protein, improve the absorption rate of the plant protein, which helps to reduce feed costs in animal feeding, ameliorate optimize growth performance and feed utilization. Lysine is required as a feed additive for domestic animal and domestic fowl, which in great demand. In the feed field, lysine as the feed additive is mostly produced by fermentation and chemical synthesis. With the chemosynthesis, large-scale production of lysine is universally obtained, but the technology was complicated, multi-product, expensive, and can not be directly added. With the microbial production, it’s more efficiency than chemical synthesis. And the production of the probiotics are green, non-toxic, non-polluting, safe and reliable, it also can be feed directly.Probiotics derived from microorganisms have been broadly applied as an additive in the food and feed industries, providing the consumer with one or more proven health benefits. Bacillus natto is a gram-positive bacterium, it’s belong to Bacillus subtilis. It has been allowed by the National Ministry of Agriculture as feed-grade microbes additives. Direct-fed Bacillus natto have been shown increasing the anminals performance, promoting growth, altering intestinal histology. Probiotics appear in alimentary duct, can increase the immunological response, feed conversion rate, antimicrobial effect and growth performance.In this experiment, I used the genetic engineering method, used PCR technique to acquire Cecropin B gene, and the recombinant plasmid p HT43/CB was constructed. Then used fusion PCR technique, to connect methionine-rich gene(zein) from maize endosperm protein with lysine-rich gene(Cflr) from the pepper anther, then the fusion gene zein-Cflr was inserted into the vector p HT43, and the recombinant plasmid p HT43/zein-Cflr was constructed. Here we developed a new protocol to prepare the Bacillus natto competent cells based on the methods of a high-efficiency and simple transformation system. After the two recombinant plasmids had been transferred into Bacillus natto, we also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. The curves of the growth of Bacillus natto and the recombinant were determined, and the experimental data showed that p HT43/CB and p HT43/zein-Cflr had almost no influence of physiological burdens on Bacillus natto.Then to verify that the Cecropin B was expressed in Bacillus natto, we detected the antibacterial activity of this natto, found that it could ameliorate the antibacterial activity of the bacteria, and the antibacterial activity against Staphylococcus aureus was better than that of both Bacillus subtilis and Lactobacillus casei.After being induced by IPTG for the expression of the fusion gene, we found that an apparent band at 40 ku site was appeared for the recombinant strain by SDS-PAGE.The contents of methionine and lysine were individually detected with HPLC,the quantities of methionine and lysine in the recombinant strain were increased by 18.37% and 24.68% than the wild one, respectively.This study provides research-basis for the application of the recombined Bacillus natto as feed additive. |
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