本文選題：假交替單胞菌 + 胞外產(chǎn)物��； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：第一部分益生菌在水產(chǎn)養(yǎng)殖中的應(yīng)用水產(chǎn)養(yǎng)殖業(yè)迅速發(fā)展,病害頻發(fā),利用益生菌來改善生態(tài)環(huán)境、預(yù)防治療水產(chǎn)動物疾病已經(jīng)越來越得到認(rèn)可。本部分對水產(chǎn)益生菌的定義、水產(chǎn)養(yǎng)殖中常用益生菌種類以及益生菌的作用機(jī)制簡單概述,并介紹了假交替單胞菌屬,該屬能夠分泌多種生物活性物質(zhì),包括小分子化合物、蛋白質(zhì)、多糖等,在水產(chǎn)病害防治、赤潮防治等方面具有較大的潛力。第二部分假交替單胞菌m8和a22胞外產(chǎn)物和胞內(nèi)產(chǎn)物抑菌活性比較本部分運(yùn)用玻璃紙覆蓋平板法制備實(shí)驗(yàn)菌株的胞外產(chǎn)物和胞內(nèi)產(chǎn)物,通過濾紙片法檢測胞外產(chǎn)物和胞內(nèi)產(chǎn)物的抑菌活性,結(jié)果表明胞外產(chǎn)物和胞內(nèi)產(chǎn)物對副溶血弧菌都有抑制作用,且胞外產(chǎn)物的抑菌效果要好于胞內(nèi)產(chǎn)物。為了優(yōu)化制備胞外產(chǎn)物的培養(yǎng)時間,分別于培養(yǎng)12h、24h、36h和48h后制備胞外產(chǎn)物,然后用濾紙片法進(jìn)行抑菌活性檢查,并對各個時間制備的胞外產(chǎn)物總蛋白含量進(jìn)行定量。結(jié)果表明,培養(yǎng)24h后制備胞外產(chǎn)物的抑菌活性最好且總蛋白含量在24h也達(dá)到最大值。第三部分假交替單胞菌m8和a22胞外產(chǎn)物相關(guān)性質(zhì)研究將過氧化氫酶濃度設(shè)為三個梯度,分別為100μg/mL,200μg/mL,500μg/mL,測試了過氧化氫酶對胞外產(chǎn)物抑菌活性的影響。結(jié)果顯示過氧化氫酶能夠抑制胞外產(chǎn)物的抑菌活性,含有過氧化氫酶的濾紙片能夠使抑菌圈產(chǎn)生缺口,但可能是濾紙片距離擺放的原因,不同濃度的過氧化氫酶產(chǎn)生缺口并沒有明顯的差異。L-氨基酸氧化酶能夠催化氨基酸釋放過氧化氫而產(chǎn)生抑菌作用,但它們的底物多種多樣。用修改后的分光光度計法檢測過氧化氫的產(chǎn)生量,將20種L-氨基酸分別作為底物,在505nm下測吸光值。結(jié)果顯示,L-賴氨酸作為底物產(chǎn)生過氧化氫的量高于其他L-氨基酸,菌株m8的胞外蛋白添加L-賴氨酸產(chǎn)生過氧化氫量高于a22,這與m8的抑菌活性好于a22相對應(yīng),推測通過產(chǎn)生過氧化氫來產(chǎn)生抑菌活性。通過變性聚丙烯酰氨凝膠電泳(SDS-PAGE)及膠內(nèi)活性檢測,發(fā)現(xiàn)分子量為70KDa的蛋白條帶具有抑菌活性,在膠條上形成明顯的抑菌條帶,并將該條帶切下來進(jìn)行質(zhì)譜分析,結(jié)果表明,菌株m8的70KDa蛋白條帶鑒定為TonB-dependent receptor[Pseudoalteromonas flavipulchra JG1],菌株a22的70KDa蛋白條帶鑒定為抗菌蛋白antibacterial protein[Pseudoalteromonas flavipulchra JG1]。第四部分菌株a22抑菌蛋白的克隆與表達(dá)根據(jù)Gene Bank公布的抑菌蛋白的序列,用PrimerPremier5.0軟件設(shè)計引物,以菌株基因組DNA為模板擴(kuò)增目的基因,將純化后的目的基因與表達(dá)載體p BAD/gⅢA連接,轉(zhuǎn)入大腸桿菌BL21,構(gòu)建重組表達(dá)菌株BL21/p BAD/gⅢA/2-p-1,在20℃用終濃度為0.5%L-阿拉伯糖進(jìn)行誘導(dǎo)表達(dá),表達(dá)產(chǎn)物經(jīng)過SDS-PAGE顯示該蛋白能在大腸桿菌中異源表達(dá),但是表達(dá)產(chǎn)物的可溶部分并未檢測到抑菌活性,推測可能是形成了包涵體,無法進(jìn)行翻譯后修飾,導(dǎo)致表達(dá)產(chǎn)物沒有抑菌活性。
[Abstract]:The first part of the application of probiotics in aquaculture aquaculture development, frequent occurrence of diseases, to improve the ecological environment of the use of probiotics, prevention and treatment of aquatic animal diseases has been increasingly recognized. This part of the definition of aquatic bacteria and probiotics, mechanism of probiotics briefly commonly used in aquaculture health benefits, and introduces false alternate Aeromonas genus, the genus can secrete a variety of bioactive substances, including small molecules, proteins, polysaccharides, disease prevention and control in aquaculture, has great potential in the aspect of red tide prevention. The second part Pseudoalteromonas M8 and A22 extracellular products and the intracellular antibacterial activity of this part of the application of glass paper cover plate prepared by the experimental strain of extracellular products and intracellular product, antibacterial activity by filter paper method for the detection of extracellular products and intracellular products. The results show that extracellular products And intracellular product on Vibrio parahaemolyticus has better antibacterial effect and inhibition of extracellular products from intracellular products. In order to develop the time to optimize the preparation of extracellular products, were cultured 12h, 24h, preparation of extracellular products of 36h and 48h, and then check the antibacterial activity by filter paper method. The total protein content of extracellular products and the preparation time quantitatively. The results showed that after 24h culture, preparation of extracellular products the best antibacterial activity and total protein content in 24h can reach the maximum. The third part Pseudoalteromonas M8 and A22 extracellular products on some properties of catalase concentration for the three gradient, respectively 100 g/mL, 200 g/mL, 500 g/mL, the effect of hydrogen peroxide on extracellular enzyme product antibacterial activity were tested. The results showed that catalase can inhibit the antibacterial activity of extracellular products, filter paper containing catalase can make the inhibition Bacteria produce ring gap, but may cause the filter paper from the display, the catalase concentration had different gap and no difference of.L- amino acid oxidase was capable of catalyzing amino acid release of hydrogen peroxide produced inhibitory effect, but their substrates varied. With modified spectrophotometric method for determination of hydrogen peroxide production, will 20 kinds of amino acids were L- as substrate, the absorbance measured at 505nm. The results showed that L- lysine as substrate to produce hydrogen peroxide was higher than that of other L- amino acids, strain M8 protein added L- lysine producing hydrogen peroxide was higher than that of A22, and the antibacterial activity of M8 is better than A22 corresponding it suggests that the production of hydrogen peroxide to produce antibacterial activity. By denaturing polyacrylamide gel electrophoresis (SDS-PAGE) detection and in gel activity, found that the molecular weight of 70KDa protein bands with antibacterial activity, in Glue to form a significant inhibition zone, and the strip cut down by mass spectrometry analysis, the results show that the strain M8 70KDa protein band was identified as TonB-dependent receptor[Pseudoalteromonas flavipulchra JG1], A22 strain 70KDa protein band was identified as antibacterial protein[Pseudoalteromonas flavipulchra JG1]. cloning antibacterial protein fourth strain A22 inhibitory protein and expression of the the sequence of Gene inhibitory protein released by Bank, primers were designed by PrimerPremier5.0 software according to the genomic DNA as template to amplify the target gene, the gene expression vector and purified P BAD/g III A connection into E.coli BL21. The recombinant expression strains of BL21/p BAD/g III A/2-p-1 at 20 DEG C, with a final concentration of 0.5%L- in Arabia sugar induced expression product by SDS-PAGE showed that the protein was heterologously expressed in Escherichia coli, However, the soluble part of the expressed product did not detect antibacterial activity. It was presumed that the inclusion body was formed and could not be modified after translation, resulting in no antibacterial activity of the expression product.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S948;S963.211

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王春迪;宋曉玲;張曉靜;張盛靜;孫新穎;劉寶彬;高文輝;黃P";;養(yǎng)殖水體中添加蠟樣芽孢桿菌PC465對凡納濱對蝦抗病力的影響[J];中國水產(chǎn)科學(xué);2016年01期

2 張盛靜;宋曉玲;趙小金;黃P";;飼料中添加益生菌對凡納濱對蝦抗感染和5種免疫基因表達(dá)的影響[J];水產(chǎn)學(xué)報;2015年06期

3 職通瑞;宋曉玲;張曉靜;張盛靜;黃P";;多抗間接ELISA方法的建立及其在海洋細(xì)菌快速檢測中的應(yīng)用[J];漁業(yè)科學(xué)進(jìn)展;2015年02期

4 孫子為;歸談純;高乃云;張艷森;朱明秋;;高級氧化技術(shù)降解水體中抗生素的研究進(jìn)展[J];四川環(huán)境;2014年05期

5 張家國;劉翠玲;;乳酸菌代替抗生素在水產(chǎn)養(yǎng)殖上的應(yīng)用[J];中國水產(chǎn);2014年07期

6 祖岫杰;李秀穎;劉慧吉;;水產(chǎn)養(yǎng)殖中抗菌藥物殘留原因及控制措施[J];現(xiàn)代農(nóng)業(yè)科技;2014年03期

7 楊移斌;夏永濤;陳新;邱軍強(qiáng);曹海鵬;胡鯤;楊先樂;;7種消毒劑對鱘4種病原菌體外抑菌效果[J];四川動物;2013年06期

8 郭慧;冼健安;畢建柱;葉超霞;王安利;;蝦類免疫因子的研究進(jìn)展[J];飼料工業(yè);2013年22期

9 劉洋;舒巧玉;樓春燕;吳湘;韓志萍;葉金云;;兩株光合細(xì)菌的分離鑒定及其水質(zhì)凈化效果研究[J];淡水漁業(yè);2013年05期

10 李永芹;許樂樂;陳克衛(wèi);;1株芽孢桿菌的篩選鑒定及其凈水效果研究[J];水生態(tài)學(xué)雜志;2013年01期

相關(guān)博士學(xué)位論文 前1條

1 于敏;金麗假交替單胞菌JG1抑菌機(jī)理研究[D];中國海洋大學(xué);2013年

相關(guān)碩士學(xué)位論文 前2條

1 職通瑞;飼料中補(bǔ)充有益菌對凡納濱對蝦抗病力及其腸道微生物組成影響的研究[D];上海海洋大學(xué);2014年

2 王樹杉;海洋有益菌JG1的分類鑒定及其抑菌活性物質(zhì)的分離純化與性質(zhì)研究[D];中國海洋大學(xué);2009年

，

本文編號：1731227