Expression Of Ascaris Suum Cecropin P1 Gene In Pichia Pastoris And Its Antimicrobial Activity

Antibacterial agents, as the important components of feed additives, have been widely used in livestock production in China. In the present feeding and management conditions for intensive livestock production, it is difficult to avoid the invasion of the foreign pathogenic microorganisms into the animal bodies from the different sources, such as feed, water and environment. In order to maintain the animal healthy growth, it is necessary to add some antibacterial agents in the feed for the prevention of animal disease. However, the type and quantity of the antimicrobial agents added in feed should be concerned since they are not only related with the animal health, but also related with the quality and biological safety of animal products. At the same time they are also involved in the natural environment and ecological balance. Therefore, the attention for the antimicrobial additive in the animal feed has been paid by the animal product producer and consumer.Therefore, to explore the new antibacterial additive with non-toxic, non-drug-residual and non-drug-resistant properties is the future development trends and practical requirements. Antimicrobial peptides (AMPs) are the kind of peptides that are produced by the immunological system of organism to defend the infection of pathogens. It is one of the important components in animal innate immunological system. Unlike traditional antibiotics, antimicrobial peptides can not induce drug resistance because of its unique antibacterial machnism, which suggests that AMPs can be a potential substitute for traditional antibiotics.AMPs can be obtained by the three different ways: isolation from organism, chemical synthesis and genetic engineering preparation. The animal endogenous antimicrobial peptides are very poor in natural resources, so it can not be abundantly obtained from animal tissue, such as small intestine. Chemical synthesis is a high-cost preparation, therefore can not be used to prepare the antimicrobial peptides at a large scale. However, the genetic engineering preparation is a low-cost production and can produce at large scale. So the genetic engineering preparation shows broad prospect in AMP production.Recently, the scientists in China pay more attention to the antibacterial peptide research and development. They did lots of researches on the activity analysis and the production of antibacterial peptides from the different species, such as insects about Musca domestica, silkworm, worms and some mammals. It showed that cecropin AD can inhibit many animal pathogen, such as porcine edema disease virus, porcine salmonella, avian escherichia coli, duck escherichia coil, brucella abortus and so on.β-defensin-2 added into weaning piglet feed can significantly reduce weaning piglet diarrhea. Researchers also found that procine intestine antibacterial peptides show the inhibition for various pathogenic bacteria, and the efficency of the inhibition was about 60-98%.So far, the antimicrobial peptides as feed antibacterial additive was mainly used in livestock production are cecropin AD-yeast and Musca domestica peptide product. Those products possess some inhibitory effect on some pathogens. However there are some disadvantages for using the exogenous antimicrobial peptides as the animal antimicrobial feed additives. Because mammalian antimicrobial peptides have the species-specificity. In this research, we mainly focus on the mammalian antimicrobial peptides. We amplified antimicrobial peptide Cecropin P1 cDNA from Ascaris in porcine intestines. Using Pichia pastoris expression system achieved its secretory expression in vitro.The primers were designed according to published cDNA sequence of Cecropin P1 and amplified cecropin P1 cDNA from Ascaris suum by RT-PCR. The Cecropin P1 gene was cloned into yeast shuttle vector pPIC9K down stream ofα-factor signal peptide sequence by SnaBⅠand NotⅠsites to generate the recombinant secretion vector pPIC9K/cecropin P1. The recombinant vector, linearized by SalⅠ, was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation. The His+-transformed methylotropic (His+, Mut+) yeast were selected using histidine-absent medium containing dextrose(MD) or methanol (MM) as the only carbon source, and then screened the recombinant GS115 with multi-copy Cecropin P1 genes by G418. The recombinant yeast encoding Cecropin P1 was identified by PCR. The results showed that Cecropin P1 gene was integrated into yeast genome. The secretory expression of Cecropin P1 was performed under the induction of methanol in shaking flask culture. The recombinant yeast can secretoryly express Cecropin P1 identified by Tricine-SDS-PAGE. The recombinant Cecropin P1 has the antibacterial activity against Escherichia coil and Staphylococcus aureus.