Cecropin AD Gene Constitutive Expression In Pichia Pastoris And Effects Of Performance Of Weaned Piglets

Cecropin AD is a hybrid peptide that exhibits greater antibacterial activity than that of the natural Cecropins. It is composed of 1-11 amino acid residues of Cecropin A and 12-37 amino acid residues of Cecropin D. The purpose of this study is to take constitutive expression method that antibacterial peptide Cecropin AD was expressed in Pichia pastoris SMD1168, explore the optimization of fermentation conditions for SMD1168, and study the effects of Cecropin AD on the performance of weaned piglets. The main contents include three parts as follows.1, Take constitutive expression method that antibacterial peptide Cecropin AD was expressed in Pichia pastoris SMD1168. Gene fragments SH encoding antimicrobial peptides Cecropin AD was synthesized by chemical methods. Gene fragment SH was inserted into the vector pGAPZa-A and named pGAPZa-ASH. Plasmid PGAPZa-ASH was converted to Pichia pastoris SMD1168 using electrotransformation. The result of Zeocion resistance screening showed that there was positive recombinant. Tricine-SDS-PAGE analysis showed that the molecular weight of peptides Cecropin AD was 3.0 kD, and the expression products had antibacterial activity.20μL spissated fermentation broth bacteriostasis was 17.5 mm, and 20μL Amp was 20.5 mm. Exogenous gene is stable in recombinant SMD1168. The growth curve of recombinant SMD1168 and SMD1168 was similar. This result meaned that insertion of Cecropin AD gene was not affect the growth of Pichia pastoris SMD1168.2, The optimization of main fermentation parameters of antimicrobial peptides Cecropin AD of Pichia pastoris SMD1168 in a small-scale.Optimize main fermentation parameters of recombinant SMD1168 were fermentation broth pH, dissolved oxygen, and feeding mode. The index was the identified supernatant protein with Tricine-SDS-PAGE analysis after fermentation. The optimized conditions of recombinant SMD1168 were pH 5.5,30%-40% dissolved oxygen and accelerant feeding. The expression of Cecropin AD is 225 mg/L fermentation culture liquid and 26% of total proteion was Cecropin AD 70% of fermentation broth was culture liquid.3, The effects of Cecropin AD on performance of weaned pigletsThree hundred crossbred weaned piglets (Duroc×Landrace×Yorkshine), with initial BW of 10 kg, were randomly divided into 5 groups. Each group had 6 replicates (pen) and each pen had 10 piglets. Piglets of the control group were fed basal dietary and the other groups were fed basal dietary with 10% Colistin Sulfate 300 mg/kg and 10% bacitracin zinc 1500 mg/kg, Cecropin AD premix 10,20 and 30 mg/kg, respectively. The experiment was lasted 32 days.The result showed that there had a trend of improve average daily gain and Feed/Gain of the piglets in the antibiotic group and three Cecropin AD groups than that of the control group, however, there were no significant different among the groups (P>0.05). The diarrhea rate of the antibiotic group and three Cecropin AD groups were significantly lower than that of the control group (P<0.05). These results indicated that Cecropin AD has similar effect on decreasing diarrhea rate of weaned piglets and the optimum level Cecropin AD in weaned piglets diet-is 10 mg/kg.