转天蚕素B及串联高Met和Lys纳豆芽孢杆菌工程菌的构建
发布时间：2018-11-11 11:01
【摘要】：抗菌肽是一种具有抗菌生物活性且多为小于10KD的分子多肽,可以直接通过诱导机体获取。其在食品、医药中被广为应用,同时也被认定是一类环保型饲料添加剂。天蚕素B(Cecropin B)是一种具有热稳定性的成熟小肽,仅含35个氨基酸,在已获得的众多抗菌肽中抗性较强。它对细菌、真菌、病毒等有着显著的抑制杀伤性。蛋氨酸是植物性蛋白中广为缺失的一种氨基酸,因而增添蛋氨酸于动物的日粮中不仅能够提高植物性蛋白的利用率,还能有效降低饲养动物所需饲料的成本。在饲料工业中,赖氨酸的需求量很大。饲料中添加的赖氨酸主要通过微生物发酵或化学合成的方法获取。化学合成赖氨酸的生产技术相对繁杂,副产物较多,不可直接用于添加,且价格昂贵。微生物发酵法生产赖氨酸的生物效率明显高于化学合成法,其中益生菌产生的赖氨酸更是绿色、无污染、安全且能够直接用作添加剂。益生菌是由一种或多种有益微生物及代谢物构成的活菌制剂,人类或动物能直接使用和喂食。纳豆芽孢杆菌隶属于益生菌,是枯草芽抱杆菌的一个亚种,已被纳入我国农业部允许使用的饲料级微生物添加剂。其作为饲料添加剂能够提高饲料转化率、促进仔猪生长、提高畜禽免疫能力和抗应激能力等。本实验利用基因工程方法,获取了天蚕素B基因(CB)、高蛋氨酸蛋白基因(zein)及高赖氨酸蛋白基因(Cflr),同时利用融合PCR技术将zein、Cflr进行了串联。将CB基因与串联的zein-Cflr基因分别连接至枯草芽孢杆菌的一种表达载体p HT43,构建出p HT43/CB和p HT43/zein-Cflr重组表达载体。之后,利用化学方法制备纳豆芽孢杆菌感受态,将两种重组载体分别转化至纳豆芽孢杆菌中,并验证重组载体在目的菌中稳定传代,发现其稳定率均为100%。为了验证此类重组载体对纳豆芽孢杆菌自身的生长代谢是否有促进或抑制作用,测定了重组菌与野生菌的生长曲线,结果表明该质粒几乎对目的菌无影响。对转入p HT43/CB的纳豆芽胞杆菌进行了抑菌活性的测定,抑制大肠杆菌的检测中,发现干酪乳杆菌的抑菌活性优于重组菌,枯草芽孢杆菌和重组菌的抑菌活性均较低,但重组菌的抑菌活性优于野生菌的抑菌活性;抑制金黄色葡萄球菌的检测中,发现重组菌的抑菌活性明显高于干酪乳杆菌、枯草芽孢杆菌及其野生菌。此外,还进行了Western-blot的验证,发现经诱导后有目的蛋白的表达。对转入p HT43/zein-Cflr的纳豆芽胞杆菌经IPTG诱导后进行了SDS-PAGE电泳分析,发现在40ku处出现了1条明显条带,该值与目的蛋白带相符。此外,还通过HPLC检测菌株发酵液中蛋氨酸及赖氨酸含量,发现重组菌的蛋氨酸与赖氨酸含量相较于野生型菌株分别提高了18.37%和24.68%。本实验能增强纳豆芽孢杆菌作为饲料添加剂的营养价值,为该重组菌作为饲料添加剂进一步的应用提供了技术基础。
[Abstract]:Antimicrobial peptide is a kind of molecular polypeptide with antibacterial biological activity and less than 10KD, which can be obtained directly by inducing organism. It is widely used in food and medicine, and is also recognized as a kind of environmental-friendly feed additive. B (Cecropin B) is a small mature peptide with thermal stability. It contains only 35 amino acids and is resistant to many antimicrobial peptides. It has a significant inhibitory effect on bacteria, fungi, viruses and so on. Methionine is a widely absent amino acid in plant proteins, so adding methionine to animal diets can not only improve the utilization of plant proteins, but also effectively reduce the feed cost of feeding animals. Lysine is in great demand in the feed industry. Lysine in feed is obtained by microbial fermentation or chemical synthesis. The production technology of chemical synthesis of lysine is relatively complicated and there are many by-products, which can not be directly used in addition, and the price is expensive. The biological efficiency of producing lysine by microbial fermentation is obviously higher than that by chemical synthesis. The lysine produced by probiotics is more green, pollution-free, safe and can be used as additive directly. Probiotics is a living preparation consisting of one or more beneficial microorganisms and metabolites, which can be used and fed directly by humans or animals. Bacillus natto, which belongs to probiotics, is a subspecies of Bacillus subtilis. As a feed additive, it can increase feed conversion rate, promote piglet growth, improve immune ability and anti-stress ability of livestock and poultry. In this experiment, we obtained the (CB), high methionine protein gene (zein) and the high lysine protein gene (Cflr), of the silkworm (CB), gene by genetic engineering method, and used the fusion PCR technique to connect the zein,Cflr. The recombinant expression vectors of p HT43/CB and p HT43/zein-Cflr were constructed by ligating the CB gene and the tandem zein-Cflr gene into one of the expression vectors of Bacillus subtilis, p HT43,. After that, the two recombinant vectors were transformed into Bacillus natto by chemical method. The stable passage of the recombinant vector in the target bacteria was verified, and the stability rate of the recombinant vector was found to be 100%. In order to verify whether the recombinant vector could promote or inhibit the growth and metabolism of Bacillus natto, the growth curves of the recombinant and wild bacteria were determined. The results showed that the plasmid had no effect on the target bacteria. The antimicrobial activity of Bacillus natto transformed into p HT43/CB was determined. In the detection of the inhibition of Escherichia coli, it was found that Lactobacillus casei had better antibacterial activity than the recombinant strain, and the bacteriostatic activity of Bacillus subtilis and Recombinant bacteria were lower. But the bacteriostatic activity of recombinant bacteria was better than that of wild bacteria. The bacteriostatic activity of recombinant bacteria was higher than that of Lactobacillus casei, Bacillus subtilis and wild bacteria. In addition, the expression of target protein was confirmed by Western-blot. The results of SDS-PAGE electrophoresis of Bacillus natto transformed into p HT43/zein-Cflr by IPTG showed that there was an obvious band in 40ku, which was consistent with the target protein band. In addition, the content of methionine and lysine in fermentation broth was detected by HPLC. The results showed that the content of methionine and lysine of recombinant strain was 18.37% and 24.68% higher than that of wild-type strain, respectively. This experiment can enhance the nutritional value of Bacillus natto as feed additive and provide a technical basis for its further application as feed additive.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q78;S816.7
本文编号：2324633
[Abstract]:Antimicrobial peptide is a kind of molecular polypeptide with antibacterial biological activity and less than 10KD, which can be obtained directly by inducing organism. It is widely used in food and medicine, and is also recognized as a kind of environmental-friendly feed additive. B (Cecropin B) is a small mature peptide with thermal stability. It contains only 35 amino acids and is resistant to many antimicrobial peptides. It has a significant inhibitory effect on bacteria, fungi, viruses and so on. Methionine is a widely absent amino acid in plant proteins, so adding methionine to animal diets can not only improve the utilization of plant proteins, but also effectively reduce the feed cost of feeding animals. Lysine is in great demand in the feed industry. Lysine in feed is obtained by microbial fermentation or chemical synthesis. The production technology of chemical synthesis of lysine is relatively complicated and there are many by-products, which can not be directly used in addition, and the price is expensive. The biological efficiency of producing lysine by microbial fermentation is obviously higher than that by chemical synthesis. The lysine produced by probiotics is more green, pollution-free, safe and can be used as additive directly. Probiotics is a living preparation consisting of one or more beneficial microorganisms and metabolites, which can be used and fed directly by humans or animals. Bacillus natto, which belongs to probiotics, is a subspecies of Bacillus subtilis. As a feed additive, it can increase feed conversion rate, promote piglet growth, improve immune ability and anti-stress ability of livestock and poultry. In this experiment, we obtained the (CB), high methionine protein gene (zein) and the high lysine protein gene (Cflr), of the silkworm (CB), gene by genetic engineering method, and used the fusion PCR technique to connect the zein,Cflr. The recombinant expression vectors of p HT43/CB and p HT43/zein-Cflr were constructed by ligating the CB gene and the tandem zein-Cflr gene into one of the expression vectors of Bacillus subtilis, p HT43,. After that, the two recombinant vectors were transformed into Bacillus natto by chemical method. The stable passage of the recombinant vector in the target bacteria was verified, and the stability rate of the recombinant vector was found to be 100%. In order to verify whether the recombinant vector could promote or inhibit the growth and metabolism of Bacillus natto, the growth curves of the recombinant and wild bacteria were determined. The results showed that the plasmid had no effect on the target bacteria. The antimicrobial activity of Bacillus natto transformed into p HT43/CB was determined. In the detection of the inhibition of Escherichia coli, it was found that Lactobacillus casei had better antibacterial activity than the recombinant strain, and the bacteriostatic activity of Bacillus subtilis and Recombinant bacteria were lower. But the bacteriostatic activity of recombinant bacteria was better than that of wild bacteria. The bacteriostatic activity of recombinant bacteria was higher than that of Lactobacillus casei, Bacillus subtilis and wild bacteria. In addition, the expression of target protein was confirmed by Western-blot. The results of SDS-PAGE electrophoresis of Bacillus natto transformed into p HT43/zein-Cflr by IPTG showed that there was an obvious band in 40ku, which was consistent with the target protein band. In addition, the content of methionine and lysine in fermentation broth was detected by HPLC. The results showed that the content of methionine and lysine of recombinant strain was 18.37% and 24.68% higher than that of wild-type strain, respectively. This experiment can enhance the nutritional value of Bacillus natto as feed additive and provide a technical basis for its further application as feed additive.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q78;S816.7
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