Cecropin AD在毕赤酵母中的组成型表达及对断奶仔猪生长性能的影响
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摘要
摘要
抗菌肽Cecropin AD是人工合成的由CecropinA的N端1-11肽段和Cecropin D的C端12-37肽段组成的杂合肽。本试验的目的是利用组成型表达方法在毕赤酵母SMD1168菌株表达抗菌肽Cecropin AD,探讨优化发酵重组SMD1168菌株的条件,并研究抗菌肽Cecropin AD对断奶仔猪生长性能的影响。主要从以下几方面进行研究：
1抗菌肽Cecropin AD在毕赤酵母中的组成型表达
利用化学方法合成编码抗菌肽Cecropin AD的基因片段SH,并进行PCR扩增。基因片段SH与表达载体pGAPZa-A经Xho I和Not I双酶切后连接构建为重组表达质粒pGAPZa-ASH。经过鉴定并测序正确后将pGAPZa-ASH电转化至毕赤酵母SMD1168中,通过Zeocin抗性筛选阳性重组毕赤酵母工程菌。Tricine-SDS-PAGE分析表明在3.0 KD处有目的条带,并且表达产物抗菌肽Cecropin AD有生物活性。20μL浓缩10倍的表达上清液对E.coli DH5α的抑菌直径可达到17.5 mm,小于20μLAmp (100μg/mL)相应的抑菌直径(20.5 mm)。外源基因在酵母中的稳定性很高。重组酵母与其受体菌的生长曲线相近。试验结果表明,用组成型表达方法在毕赤酵母菌株成功表达了抗菌肽Cecropin AD并且其基因片段SH的插入对酵母菌的生长规律影响不显著。
2抗菌肽Cecropin AD小规模发酵主要参数的优化
按表达手册进行表达试验,从发酵液pH、溶氧和补料等方面优化重组毕赤酵母SMD1168的主要发酵参数。采用优化的发酵参数进行发酵,对上清液TCA沉淀纯化后以Tricine-SDS-PAGE鉴定。优化后的重组毕赤酵母SMD1168发酵主要参数：发酵液pH5.5,溶氧30%-40%,补料采用变速流加方式。发酵周期为60 h,上清液中Cecropin AD的表达量为225 mg/L, Cecropin AD占表达总蛋白量的26%。发酵液离心后的上清液占总发酵液体积的70%。
3抗菌肽Cecropin AD对断奶仔猪生长性能的影响
试验选取体重约10kg的杜×大×长三元杂交断奶仔猪300头,采用单因子完全随机区组设计,分为对照组、抗生素组、试验1组、试验2组、试验3组,每个处理6个重复,每个重复10头猪,研究毕赤酵母组成型表达的抗菌肽对断奶仔猪生长性能的影响。对照组饲喂基础日粮,抗生素组在基础日粮中添加10%的硫酸抗敌素300 mg/kg和10%的杆菌肽锌1500 mg/kg,试验1组、试验2组、试验3组分别在基础日粮中添加抗菌肽Cecropin AD 10 mg/kg、20 mg/kg、30 mg/kg。试验结果表明,与对照组相比,日粮中添加抗生素或抗菌肽有提高断奶仔猪日增重和饲料转化效率的趋势,但没达到显著差异(P>0.05)；试验各组仔猪的腹泻率均显著低于对照组(P<0.05),表明抗菌肽CecropinAD可以达到与抗生素类似的防腹泻效果。综合考虑仔猪的生长性能和腹泻指数,以添力口10 mg/kg Cecropin AD效果最佳。
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 piglets
Three 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.
抗菌肽Cecropin AD是人工合成的由CecropinA的N端1-11肽段和Cecropin D的C端12-37肽段组成的杂合肽。本试验的目的是利用组成型表达方法在毕赤酵母SMD1168菌株表达抗菌肽Cecropin AD,探讨优化发酵重组SMD1168菌株的条件,并研究抗菌肽Cecropin AD对断奶仔猪生长性能的影响。主要从以下几方面进行研究：
1抗菌肽Cecropin AD在毕赤酵母中的组成型表达
利用化学方法合成编码抗菌肽Cecropin AD的基因片段SH,并进行PCR扩增。基因片段SH与表达载体pGAPZa-A经Xho I和Not I双酶切后连接构建为重组表达质粒pGAPZa-ASH。经过鉴定并测序正确后将pGAPZa-ASH电转化至毕赤酵母SMD1168中,通过Zeocin抗性筛选阳性重组毕赤酵母工程菌。Tricine-SDS-PAGE分析表明在3.0 KD处有目的条带,并且表达产物抗菌肽Cecropin AD有生物活性。20μL浓缩10倍的表达上清液对E.coli DH5α的抑菌直径可达到17.5 mm,小于20μLAmp (100μg/mL)相应的抑菌直径(20.5 mm)。外源基因在酵母中的稳定性很高。重组酵母与其受体菌的生长曲线相近。试验结果表明,用组成型表达方法在毕赤酵母菌株成功表达了抗菌肽Cecropin AD并且其基因片段SH的插入对酵母菌的生长规律影响不显著。
2抗菌肽Cecropin AD小规模发酵主要参数的优化
按表达手册进行表达试验,从发酵液pH、溶氧和补料等方面优化重组毕赤酵母SMD1168的主要发酵参数。采用优化的发酵参数进行发酵,对上清液TCA沉淀纯化后以Tricine-SDS-PAGE鉴定。优化后的重组毕赤酵母SMD1168发酵主要参数：发酵液pH5.5,溶氧30%-40%,补料采用变速流加方式。发酵周期为60 h,上清液中Cecropin AD的表达量为225 mg/L, Cecropin AD占表达总蛋白量的26%。发酵液离心后的上清液占总发酵液体积的70%。
3抗菌肽Cecropin AD对断奶仔猪生长性能的影响
试验选取体重约10kg的杜×大×长三元杂交断奶仔猪300头,采用单因子完全随机区组设计,分为对照组、抗生素组、试验1组、试验2组、试验3组,每个处理6个重复,每个重复10头猪,研究毕赤酵母组成型表达的抗菌肽对断奶仔猪生长性能的影响。对照组饲喂基础日粮,抗生素组在基础日粮中添加10%的硫酸抗敌素300 mg/kg和10%的杆菌肽锌1500 mg/kg,试验1组、试验2组、试验3组分别在基础日粮中添加抗菌肽Cecropin AD 10 mg/kg、20 mg/kg、30 mg/kg。试验结果表明,与对照组相比,日粮中添加抗生素或抗菌肽有提高断奶仔猪日增重和饲料转化效率的趋势,但没达到显著差异(P>0.05)；试验各组仔猪的腹泻率均显著低于对照组(P<0.05),表明抗菌肽CecropinAD可以达到与抗生素类似的防腹泻效果。综合考虑仔猪的生长性能和腹泻指数,以添力口10 mg/kg Cecropin AD效果最佳。
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 piglets
Three 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.
引文
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