本文選題：噬菌體內(nèi)溶素　切入點(diǎn)：E基因　出處：《內(nèi)蒙古工業(yè)大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：隨著致病性細(xì)菌耐藥性的加劇,尋找有效且不易造成菌株耐藥性的抗菌制劑迫在眉睫。噬菌體以及噬菌體編碼的內(nèi)溶素,是治療和預(yù)防細(xì)菌感染最為理想的抗生素替代物,且它們對(duì)抗耐藥性細(xì)菌具有較強(qiáng)的殺菌性。所以,從噬菌體中篩選具有抗菌活性的蛋白或多肽是一種新的途徑和方法。 本研究首先從雞糞中分離到一株烈性噬菌體,編碼該噬菌體衣殼蛋白的23基因序列與大腸桿菌噬菌體RB69的23基因序列(NCBI登錄號(hào):AY303349.1)的相似度達(dá)96%,命名為大腸桿菌噬菌體WL08;并從該病毒中克隆編碼內(nèi)溶素的E基因,序列分析結(jié)果表明,E基因全長(zhǎng)474bp,編碼157個(gè)氨基酸,與大腸桿菌噬菌體RB69的E基因相似性達(dá)98%;隨后對(duì)E基因進(jìn)行改造,用三個(gè)(Gly-Gly-Gly-Gly-Ser)單元構(gòu)成的橋,將RI基因和E基因橋連,獲得RIE基因,PCR的方法將E基因分三段擴(kuò)增:E_(1-87)、E_(34-157)和E_(110-157)。 將RIE、E、E_(1-87)、E_(34-157)和E_(110-157)基因克隆到pHERD30T載體上,通過(guò)生長(zhǎng)曲線(xiàn)的測(cè)定,初步確定E蛋白和改造后的RIE蛋白、E_(1-87)蛋白、E_(34-157)蛋白和E_(110-157)蛋白對(duì)大腸桿菌的抑制作用;PCR擴(kuò)增E基因,克隆到pTYB11載體,并轉(zhuǎn)化到大腸桿菌ER2566感受態(tài)細(xì)胞中,誘導(dǎo)表達(dá)E蛋白。初步測(cè)定了E蛋白純品的體外抑菌活性,實(shí)驗(yàn)結(jié)果顯示,在E蛋白終濃度達(dá)到1mg/ml時(shí),E蛋白對(duì)大腸桿菌Top10和金黃色葡萄球菌兩株菌都有一定的抑制作用。
[Abstract]:With the aggravation of the drug resistance of pathogenic bacteria, it is urgent to find effective antimicrobial agents that are not easy to cause bacterial resistance. Bacteriophages and endolysin encoded by bacteriophages are the most ideal antibiotic substitutes for the treatment and prevention of bacterial infections. Therefore, screening proteins or peptides with antibacterial activity from phages is a new way and method. In this study, a strong phage was isolated from chicken manure. The sequence of 23 gene encoding this phage capsid protein is similar to that of Escherichia coli phage RB69. The similarity between the gene sequence and the accession number of Escherichia coli phage RB69 is 96g, named as Escherichia coli bacteriophage WL08, and the E gene encoding endolysin is cloned from the virus. Sequence analysis showed that the E gene encoding 157 amino acids was similar to the E gene of Escherichia coli bacteriophage RB69, and then the E gene was modified by using three Gly-Gly-Gly-Gly-Gly-Gly-Serial units, and the RI gene and E gene were bridged. The E gene was amplified in three parts by using RIE gene. The E gene was amplified in three parts. The gene was cloned into the pHERD30T vector. The E protein E and the modified RIE protein Estax 34-157) were preliminarily determined by the determination of the growth curve. The inhibitory effect of E protein on Escherichia coli was amplified and cloned into pTYB11 vector. It was transformed into Escherichia coli ER2566 cells and induced to express E protein. The bacteriostatic activity of pure E protein was determined in vitro, and the results showed that, When the final concentration of E protein reached 1 mg / ml, the E protein could inhibit both Escherichia coli Top10 and Staphylococcus aureus strains.
【學(xué)位授予單位】：內(nèi)蒙古工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R341

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