日本沼虾肌抑素前体肽在枯草芽孢杆菌的表达及其对生长效果的影响

THE EXPRESSION OF MYOSTATIN PROPETIDE IN BACILLUS SUBTILIS AND ITS EFFECT ON THE GROWTH OF MACROBRACHIUM NIPPONENESE

为研究日本沼虾肌抑素前体肽(MSTNpp)对生长的作用机理,实验采用枯草芽孢杆菌表达日本沼虾肌抑素前体肽蛋白,按照枯草芽孢杆菌的偏爱密码子,对MSTNpp基因序列进行优化合成BsMSTNpp,构建得到重组表达质粒pGJ105-BsMSTNpp。经枯草芽孢杆菌转化、发酵及Western blot鉴定,结果显示重组MSTNpp蛋白分子量约为36.0 kD。重组MSTNpp表达量随发酵时间延长逐渐升高,100h表达量相对于24h提高了10倍。为验证表达产物的生物活性,选择平均体重为(1.52±0.11)g,平均体长为(4.55±0.21)cm的健康日本沼虾,随机分为4组,每组3个重复,每个重复200尾,在日本沼虾基础饲料中添加0.5‰和1‰重组枯草芽孢杆菌菌剂(109 CFU/g),作为实验组1和实验组2;投喂日本沼虾基础饲料,作为对照组1,在日本沼虾基础饲料中添加1‰空白枯草芽孢杆菌,作为对照组2;试验周期30d。结果显示,实验组1、实验组2日本沼虾特定生长率显著高于对照组1(P<0.05),和对照组2间不存在显著性差异;实验组1、实验组2的肌酸激酶(Creatine kinase,CK)活性显著高于对照组(P<0.05)。结果表明,重组表达MSTNpp枯草芽孢杆菌能有效提高日本沼虾CK酶活性,推测MSTNpp通过促进肌细胞增殖和分化提高肌肉组织生长,从而进一步影响日本沼虾生长速度。实验结果可以为进一步研究肌抑素前体肽应用于日本沼虾养殖提供技术支撑。

Myostatin propetide(MSTNpp)of Macrobrachium nipponenese was expressed in Bacillus subtilis and its effect on the growth and creatine kinase activity was investigated.The MSTNpp gene sequence was optimized to synthesize BsMSTNpp according to the B.subtilis preference codon,and the recombinant expression plasmid pGJ105-BsMSTNpp was obtained.After the transformation and fermentation of recombinant B.subtilis,the supernatant was collected and identified by Western blot.The results showed that the molecular weight of the recombinant protein was 36.0 kD.The level of recombinant BsMSTNpp increased over time,and the expression level at 100h was 10 times than that in 24h.To verify the biological activity of the recombinant BsMSTNpp,the healthy freshwater shrimp with an average body weight of 1.52 g and average body length of 4.55 cm were randomly divided into 4 groups with 3 replicates per group and 200 shrimps per replicate for a 30d trial.The experimental group 1 and experimental group 2 were fed the basal diet supplemented with recombinant pGJ105-BsMSTNpp at the dose of 0.5×10^6 CFU/g and 1.0×10^6 CFU/g respectively.The control group 1 was fed the basal diet,and the control group 2 was fed the basal diet with pGJ105 at the dose of 1.0×10^6 CFU/g.The results showed that the growth ratios of the experimental groups were significantly higher than that of the control group 1(P<0.05),and there was no significant difference between experimental groups and the control group 2.The creatine kinase activities of the experimental groups were higher than those of control groups(P<0.05).The results revealed that recombinant BsMSTNpp could enhance the activity of creatine kinase to improve the myocyte proliferation and differentiation to increase the muscle growth rate of Macrobrachium nipponenese.The results provide technical support to study the function of MSTNpp and its application in shrimp aquaculture.