| Many small peptides exert immunomodulating activities in recent reports. Called as immunopeptide, they interact in vitro with various cell populations of the immune system, such as stimulated phagocytic activity of macrophages and expression of differentiation antigens on T and B cells. In vivo, they enhance or depress, according to the case, cell-mediated and humoral immune functions, improve the ability of resisted pathogeny infection.The immunostimulating hybrid peptide was designed with a chymotrypsin digestion site and eight immunopeptides, such as Val-Glu-Pro-Ile-Pro-Tyr, Gly-Leu-Phe, etc. The start codon ATG and stop codon TAA were respectively added to the C-end and the N-end of the hybrid peptide to express it accurately. To determine the expression of the small peptide and separate it easily, an N-terminal HiS6-tag downstream of the start codon was designed. The coding sequence of immunopeptide, named as IMP, was designed according to the preferred codons of yeast saccharomyces cerevisiae.The DNA sequence coding for the IMP was synthesized in two parts. A complete small peptide coding sequence was obtained by PCR implification, EcoR I digestion and ligation. Then the full-lengthed DNA sequence was cloned at the Bgl II site into the expression vector pMA91, under the control of the PGK promoter. Then the recombinant plasmid pMA91-IMP was transformed into E. coli JM109. To confirm the correct construct, the isolated plasmid was verified by restriction enzyme analyzing and sequencing.The resulting vector, pMA91-IMP, was transformed into the yeast S. cerevisiae H158 by LiAc/ssDNA/PEG method. The transformants were selected in YNB medium plate lacking leucine, and confirmed by PCR. The small peptides with His-tag were produced in 5. Cerevisiae, and then extracted with Ni-NTA His·Bind Resin and determined by SDS-PAGE assay.Some factors such as initial pH, glucose concentration, cultivate time, and temperature were investigated to optimize the cultivation condition in the shake culture, and obtained the best fermentation medium formula of the strain H158 (pMA91-IMP). Under optimized condition, the extend cultivation was carried in 10 L fermentation tank.The effect of transformed yeast was performed with the larvae of Scophthatmus maximus L. The first phase is the 6-20th days after hatching. In this phase, the larvae switch from endogenous to exogenous nutrition. It is crucial moment of feeding management, for the high death rate of larvae. The experiment larvae were fed with the rotifers which were enriched with transformed yeast. The second phase, after 20th day, the transformed yeast was fed as food additives.After two months feeding, the anti-disease ability, survive rate and body length of experiment juveniles were obviously enhanced in contrast to the control. The body of experiment juveniles was about 7.0 mm longer than the control on average. The growth of experiment juveniles was natural, and the incidence of disease was lower than the control. In the later of experiment, the albinism occurred in the control juveniles, and induced some Scophthatmus maximus L. juveniles died. Therefore, it was implied that the transformed immunomodulating peptides yeast could improve the immunity of larvae albinism and elevate the survive rate in fry breeding.
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