本文選題：抗菌肽 + 融合表達(dá)��； 參考：《陜西科技大學(xué)》2017年碩士論文

【摘要】：抗菌肽作為生物免疫系統(tǒng)的主要組成部分,是生物體的免疫作用產(chǎn)生的抗菌譜廣、難以產(chǎn)生耐藥性的一類帶正電荷的兩親性小分子多肽。目前在醫(yī)藥、食品及畜牧業(yè)領(lǐng)域應(yīng)用廣泛。將兩種或多種抗菌肽片段雜合形成一種新型的雜合肽,能夠彌補(bǔ)天然抗菌肽的缺點(diǎn),增加抗菌肽的抑菌活性。本研究將蛙皮素抗菌肽與死亡素抗菌肽的有效作用部位拼接,獲得新型的雜合肽MT。根據(jù)雜合抗菌肽MT基因與大腸桿菌密碼子的偏好性設(shè)計(jì)了該雜合肽的基因序列,對(duì)該基因進(jìn)行生物信息學(xué)分析;利用SOE-PCR技術(shù)設(shè)計(jì)了三段引物,經(jīng)過兩輪PCR擴(kuò)增獲得目的基因MT,將目的基因克隆至大腸桿菌表達(dá)載體pGEX-6P-1構(gòu)建重組表達(dá)載體pGEX-6P-1-MT;將構(gòu)建的重組表達(dá)載體轉(zhuǎn)化到表達(dá)菌株E.coli BL21(DE3)中構(gòu)建了基因工程菌pGEX-6P-1-MT/BL21(DE3),通過對(duì)IPTG濃度、誘導(dǎo)時(shí)間、溫度以及培養(yǎng)基等發(fā)酵條件的優(yōu)化,利用GST瓊脂糖凝膠層析柱對(duì)融合蛋白進(jìn)行純化,腸激酶裂解后對(duì)雜合抗菌肽的抑菌活性進(jìn)行初步分析。本論文研究結(jié)果如下:(1)生物信息學(xué)手段對(duì)雜合抗菌肽MT的理化參數(shù)、二級(jí)結(jié)構(gòu)、親水性等進(jìn)行預(yù)測(cè)得出:氨基酸總數(shù)為29,分子量大小為3.35 kDa,等電點(diǎn)pI為10.57,含有α螺旋與β折疊結(jié)構(gòu),親水性圖譜結(jié)果顯示該雜合肽具有兩親性,具有抗菌潛力。根據(jù)大腸桿菌密碼子偏好性,優(yōu)化了該基因序列,最終得到具有抗菌潛力的目的基因MT序列。(2)利用重疊延伸擴(kuò)增(SOE-PCR)技術(shù),用合成的三段引物通過兩輪PCR成功合成了雜合抗菌肽MT的基因。(3)目的基因MT與表達(dá)載體p GEX-6P-1用EcoR I和BamH I雙酶切,然后進(jìn)行重組載體的構(gòu)建,成功獲得了重組表達(dá)載體pGEX-6P-1-MT。(4)超聲破碎細(xì)胞進(jìn)行SDS-PAGE電泳發(fā)現(xiàn),融合蛋白以可溶形式存在于菌體上清,Western-blot證明目的蛋白表達(dá),用GST瓊脂糖凝膠純化柱對(duì)雜合肽純化,純化后獲得融合蛋白,占菌體總量的50.1%,蛋白的表達(dá)量為49.5 mg/L,腸激酶切割后得到分子量為3.35 kDa大小的MT目的蛋白。(5)通過對(duì)雜合抗菌肽MT工程菌誘導(dǎo)表達(dá)條件的優(yōu)化,獲得了雜合抗菌肽MT工程菌的優(yōu)化后條件分別為:IPTG濃度0.8 mM;誘導(dǎo)時(shí)間3 h;誘導(dǎo)溫度37℃;TB培養(yǎng)基。(6)采用抑菌圈法對(duì)雜合抗菌肽MT的抑菌活性進(jìn)行研究,發(fā)現(xiàn)該雜合肽對(duì)大腸桿菌DH5α、金黃色葡萄球菌、枯草芽孢桿菌均有抑制作用,抑菌圈大小分別為5 mm、6 mm和10 mm,對(duì)枯草芽孢桿菌的抑制效果強(qiáng)于前兩者。最小抑菌濃度分別20μM、16.5μM、9μM。本研究成功實(shí)現(xiàn)了雜合肽MT在大腸桿菌pGEX-6P-1表達(dá)載體中的可溶性表達(dá),通過純化獲得了純度較高、具有抗菌活性的MT目的蛋白,在食品、醫(yī)療、飼料領(lǐng)域等有一定的應(yīng)用價(jià)值。
[Abstract]:As the main component of biological immune system, antimicrobial peptide is a kind of amphiphilic polypeptide with positive charge which is difficult to produce due to its wide spectrum of antimicrobial activity. It is widely used in medicine, food and animal husbandry. Two or more antimicrobial peptides were hybridized to form a new hybrid peptide, which can make up for the shortcomings of natural antimicrobial peptides and increase the antibacterial activity of antimicrobial peptides. In this study, a novel hybrid peptide MTP was obtained by splicing the effective sites of the antimicrobial peptide of frog skin and the antibacteriopeptide of death. According to the preference between the MT gene and the codon of Escherichia coli, the gene sequence of the hybrid peptide was designed, and the gene was analyzed by bioinformatics, and a three-piece primer was designed by using SOE-PCR technique. After two rounds of PCR amplification, the target gene was cloned into E. coli expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-MTand the recombinant expression vector was transformed into the expression strain E.coli BL21DE3. The optimal fermentation conditions, such as induction time, temperature and culture medium, were optimized. The fusion protein was purified by GST agarose gel chromatography and the antibacterial activity of the hybrid antibacterial peptide was preliminarily analyzed after the decomposition of enterokinase. The results of this study are as follows: (1) Physicochemical parameters, secondary structure of hybrid antimicrobial peptide MT by bioinformatics, The hydrophilic prediction showed that the total number of amino acids was 29, the molecular weight was 3.35 kDa, the isoelectric point Pi was 10.57, and had 偽 helix and 尾 folding structure. The hydrophilic map showed that the hybrid peptide had amphiphilic properties and antibacterial potential. According to the preference of Escherichia coli codon, the gene sequence was optimized, and the target gene MT sequence with antibacterial potential was obtained. The target gene MT and the expression vector p GEX-6P-1 were digested with EcoR I and BamH I by two rounds of PCR, and the recombinant vector was constructed. The recombinant expression vector pGEX-6P-1-MT.f4 was successfully obtained by SDS-PAGE electrophoresis. It was found that the fusion protein was expressed in the supernatant of the cell in Western-blot. The fusion protein was purified by GST agarose gel column. After purification, the fusion protein was obtained, accounting for 50.1% of the total mycelium. The protein expression was 49.5 mg / L, and the target protein of MT with molecular weight of 3.35 kDa was obtained after enterokinase cleavage. The optimized conditions of the engineering strain of hybrid antimicrobial peptide MT were obtained as follows: concentration of 1: IPTG 0.8 mm; induction time 3 h; induction temperature 37 鈩，

本文編號(hào)：1967003