【摘要】：抗菌肽是一種具有抗菌生物活性且多為小于10KD的分子多肽,可以直接通過誘導(dǎo)機(jī)體獲取。其在食品、醫(yī)藥中被廣為應(yīng)用,同時也被認(rèn)定是一類環(huán)保型飼料添加劑。天蠶素B(Cecropin B)是一種具有熱穩(wěn)定性的成熟小肽,僅含35個氨基酸,在已獲得的眾多抗菌肽中抗性較強(qiáng)。它對細(xì)菌、真菌、病毒等有著顯著的抑制殺傷性。蛋氨酸是植物性蛋白中廣為缺失的一種氨基酸,因而增添蛋氨酸于動物的日糧中不僅能夠提高植物性蛋白的利用率,還能有效降低飼養(yǎng)動物所需飼料的成本。在飼料工業(yè)中,賴氨酸的需求量很大。飼料中添加的賴氨酸主要通過微生物發(fā)酵或化學(xué)合成的方法獲取�；瘜W(xué)合成賴氨酸的生產(chǎn)技術(shù)相對繁雜,副產(chǎn)物較多,不可直接用于添加,且價格昂貴。微生物發(fā)酵法生產(chǎn)賴氨酸的生物效率明顯高于化學(xué)合成法,其中益生菌產(chǎn)生的賴氨酸更是綠色、無污染、安全且能夠直接用作添加劑。益生菌是由一種或多種有益微生物及代謝物構(gòu)成的活菌制劑,人類或動物能直接使用和喂食。納豆芽孢桿菌隸屬于益生菌,是枯草芽抱桿菌的一個亞種,已被納入我國農(nóng)業(yè)部允許使用的飼料級微生物添加劑。其作為飼料添加劑能夠提高飼料轉(zhuǎn)化率、促進(jìn)仔豬生長、提高畜禽免疫能力和抗應(yīng)激能力等。本實驗利用基因工程方法,獲取了天蠶素B基因(CB)、高蛋氨酸蛋白基因(zein)及高賴氨酸蛋白基因(Cflr),同時利用融合PCR技術(shù)將zein、Cflr進(jìn)行了串聯(lián)。將CB基因與串聯(lián)的zein-Cflr基因分別連接至枯草芽孢桿菌的一種表達(dá)載體p HT43,構(gòu)建出p HT43/CB和p HT43/zein-Cflr重組表達(dá)載體。之后,利用化學(xué)方法制備納豆芽孢桿菌感受態(tài),將兩種重組載體分別轉(zhuǎn)化至納豆芽孢桿菌中,并驗證重組載體在目的菌中穩(wěn)定傳代,發(fā)現(xiàn)其穩(wěn)定率均為100%。為了驗證此類重組載體對納豆芽孢桿菌自身的生長代謝是否有促進(jìn)或抑制作用,測定了重組菌與野生菌的生長曲線,結(jié)果表明該質(zhì)粒幾乎對目的菌無影響。對轉(zhuǎn)入p HT43/CB的納豆芽胞桿菌進(jìn)行了抑菌活性的測定,抑制大腸桿菌的檢測中,發(fā)現(xiàn)干酪乳桿菌的抑菌活性優(yōu)于重組菌,枯草芽孢桿菌和重組菌的抑菌活性均較低,但重組菌的抑菌活性優(yōu)于野生菌的抑菌活性;抑制金黃色葡萄球菌的檢測中,發(fā)現(xiàn)重組菌的抑菌活性明顯高于干酪乳桿菌、枯草芽孢桿菌及其野生菌。此外,還進(jìn)行了Western-blot的驗證,發(fā)現(xiàn)經(jīng)誘導(dǎo)后有目的蛋白的表達(dá)。對轉(zhuǎn)入p HT43/zein-Cflr的納豆芽胞桿菌經(jīng)IPTG誘導(dǎo)后進(jìn)行了SDS-PAGE電泳分析,發(fā)現(xiàn)在40ku處出現(xiàn)了1條明顯條帶,該值與目的蛋白帶相符。此外,還通過HPLC檢測菌株發(fā)酵液中蛋氨酸及賴氨酸含量,發(fā)現(xiàn)重組菌的蛋氨酸與賴氨酸含量相較于野生型菌株分別提高了18.37%和24.68%。本實驗?zāi)茉鰪?qiáng)納豆芽孢桿菌作為飼料添加劑的營養(yǎng)價值,為該重組菌作為飼料添加劑進(jìn)一步的應(yīng)用提供了技術(shù)基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q78;S816.7

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