本文關(guān)鍵詞： 抗生素 唾液乳桿菌SJH-8 細(xì)菌素 生物學(xué)特性 純化 氨基酸序列　出處：《武漢紡織大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：抗生素的使用,為人類(lèi)、動(dòng)植物預(yù)防及治療病菌感染等疾病做出了重要貢獻(xiàn),但由于抗生素難降解,使環(huán)境遭到嚴(yán)重破壞與污染,且已誘發(fā)多種抗性基因的產(chǎn)生,人類(lèi)的身體健康受到嚴(yán)重的威脅。乳酸菌素作為一種多肽或蛋白類(lèi)物質(zhì),可被酶水解,是一種環(huán)境友好型抗菌物質(zhì),具有替代抗生素使用的潛力。我國(guó)乳酸菌資源豐富,研究穩(wěn)定、高效、廣譜、無(wú)抗性的乳酸菌素近年來(lái)成為熱點(diǎn)。研究多以分離純化、生物學(xué)特性、作用機(jī)理為主,而對(duì)其氨基酸序列及基因序列等信息獲得較少,難以對(duì)其進(jìn)行量化,從而為其應(yīng)用帶來(lái)一定的困難。因此,本課題著手于開(kāi)發(fā)一種新型的乳酸菌素,并研究其氨基酸序列及基因序列,為今后的異源表達(dá)構(gòu)建重組工程菌株來(lái)生產(chǎn)細(xì)菌素提供有力的前提條件。本課題首先從珠江口水產(chǎn)動(dòng)物腸道中分離乳酸菌,以副溶血弧菌(Vibro parahaemolyticus ATCC 17802)和金黃色葡萄球菌(Staphylococcus aureus ATCC35844)為指示菌,以篩選具有廣譜抑菌活性的菌株。然后對(duì)該乳酸菌所產(chǎn)細(xì)菌素的生物學(xué)特性進(jìn)行研究,并分離純化該細(xì)菌素,后通過(guò)SDS-PAGE及MALDI-TOF/TOF質(zhì)譜檢測(cè)推測(cè)出細(xì)菌素的氨基酸序列,并以特異引物擴(kuò)增出表達(dá)該細(xì)菌素的基因,為細(xì)菌素替代抗生素的應(yīng)用提供依據(jù),并為其生產(chǎn)提供前提條件。具體研究結(jié)果如下:1.從珠江口水產(chǎn)動(dòng)物健康羅非魚(yú)、白斑魚(yú)、南美白對(duì)蝦腸道中共篩選出97株乳酸菌,以V.parahaemolyticus和S.aureus為指示菌、用濾紙片瓊脂擴(kuò)散法做抑菌活性初篩和復(fù)篩后,獲得4株對(duì)兩種指示菌均有抑菌活性的乳酸菌,其中菌株SJH-8抑菌活性最好,因此選用該菌作為研究細(xì)菌素的供試菌。基于菌株的16S r DNA序列構(gòu)建系統(tǒng)發(fā)育樹(shù),確定SJH-8為一株唾液乳桿菌。2.經(jīng)硫酸銨鹽析后對(duì)陰性菌有抑制作用的活性部分仍處于上清中,而沉淀樣品對(duì)多種革蘭氏陽(yáng)性致病菌有較強(qiáng)的抑菌活性,對(duì)陰性菌無(wú)抑菌活性。沉淀樣品中的活性成分可被胰蛋白酶完全水解,經(jīng)木瓜蛋白酶、胃蛋白酶、蛋白酶K處理后活性有所減弱;有較強(qiáng)的熱穩(wěn)定和p H穩(wěn)定性,于121℃下處理15 min或p H2.0和p H10.0處理2 h,仍保留較好活性。3.經(jīng)SDS-PAGE和MALDI-TOF/TOF質(zhì)譜檢測(cè),推測(cè)出了細(xì)菌素SJH-8的部分氨基酸序列,并確定該細(xì)菌素是由SJH-8α和SJH-8β兩條多肽組成的ClassⅡb類(lèi)肽。后根據(jù)基因abp118設(shè)計(jì)特異引物,以基因SJH-8的DNA為模板進(jìn)行PCR擴(kuò)增,最后得到表達(dá)細(xì)菌素的基因序列。
[Abstract]:The use of antibiotics has made important contributions to the prevention and treatment of human, animal and plant diseases such as bacterial infections. However, due to the difficult degradation of antibiotics, the environment has been seriously damaged and polluted, and many resistance genes have been induced. As a kind of polypeptide or protein substance, lactic acid bacteriocin can be hydrolyzed by enzyme, it is a kind of environment-friendly antimicrobial substance and has the potential to replace antibiotics. The study of stable, efficient, broad-spectrum, non-resistant lactic acid bacillins has become a hot topic in recent years. Most of the studies focus on isolation and purification, biological characteristics, action mechanism, but less information on amino acid sequence and gene sequence. It is difficult to quantify it, which brings some difficulties to its application. Therefore, this paper begins to develop a new type of lactic acid bacteriocin, and study its amino acid sequence and gene sequence. In order to construct the recombinant engineering strain to produce bacteriocin in the future, we first isolated lactic acid bacteria from the aquatic animal intestine of the Pearl River Estuary. Vibro-Vibro parahaemolyticus ATCC 17802) and Staphylococcus aureus ATCC35844) were used to screen the strains with broad-spectrum antibacterial activity. The biological characteristics of bacteriocin produced by the Lactobacillus were studied, and the bacteriocin was isolated and purified. The amino acid sequence of bacteriocin was detected by SDS-PAGE and MALDI-TOF/TOF mass spectrometry, and the gene expressing bacteriocin was amplified with specific primers, which provided the basis for the application of bacteriocin in the substitution of antibiotics. The specific results are as follows: 1.A total of 97 strains of lactic acid bacteria were screened from the intestines of healthy tilapia, white spotted fish and Penaeus vannamei in the Pearl River Estuary, with V. parahaemolyticus and S. aureus as indicators. Four lactic acid bacteria with antimicrobial activity were obtained by using filter paper Agar diffusion method, and four strains of lactic acid bacteria were obtained after screening and rescreening. Among them, the strain SJH-8 had the best bacteriostatic activity. The phylogenetic tree was constructed based on the 16s r DNA sequence of the strain, and SJH-8 was identified as a Lactobacillus saliva strain. The inhibitory activity of SJH-8 on negative bacteria was still in the supernatant after ammonium sulfate salting out. However, the precipitated samples have strong bacteriostatic activity against various gram-positive pathogens, but no inhibitory activity against negative bacteria. The active components in the precipitated samples can be completely hydrolyzed by trypsin, and can be hydrolyzed by papain, pepsin, pepsin, pepsin, papain, pepsin, papain, pepsin, pepsin. The activity of protease K was weakened after treatment with protease K, and had strong thermal stability and pH stability. After being treated at 121 鈩，

本文編號(hào)：1538968