本文選題：抗菌肽 + 天蠶素B�。� 參考：《西北大學(xué)》2005年碩士論文

【摘要】：抗菌肽(anti-bacterial peptides,簡稱ABP)是生物細胞特定基因編碼產(chǎn)生的一類小分子多肽,是宿主防御病原微生物入侵的重要分子屏障,其生成和釋放是機體炎癥反應(yīng)的組成部分�？咕牟粌H對革蘭氏陰性細菌和革蘭氏陽性細菌具有高效廣譜的殺滅能力,而且對某些病毒、真菌和原蟲也具有抑殺作用;同時它對一些腫瘤細胞具有選擇性殺傷作用,但對正常哺乳動物和昆蟲細胞卻無明顯的毒副作用。人溶菌酶(human lysozyme)具有抗革蘭氏陰性細菌和抗一些病毒的能力。人溶菌酶是人體內(nèi)的一種蛋白質(zhì),和人體具有天然相容性。在臨床上應(yīng)用時,比其它溶菌酶更安全,沒有刺激性和副作用。本實驗?zāi)康氖窍胪ㄟ^基因重組,以融合的形式將Cecropin B和人溶菌酶基因連接至高效的大腸桿菌表達載體上并進行表達,進一步探討融合蛋白的抗菌和抗病毒活性,以期研制出具有更高活性的抗菌和抗病毒重組蛋白。 為了構(gòu)建抗菌肽B(Cecropin B)和人溶菌酶(hLyso)的基因克隆載體,通過重疊區(qū)擴增法人工合成抗菌肽B基因,從pUC118-hLvso上卸下人溶菌酶基因,然后按照正確的閱讀框架融合并重組至克隆載體中。通過對重組質(zhì)粒的測序,表明融合基因Cecropin B-human Lysozyme已經(jīng)正確克隆至pBS-T載體中,將重組質(zhì)粒轉(zhuǎn)入大腸桿菌里,使目的基因能夠在大腸桿菌里保存并且大量擴增,為構(gòu)建表達載體表達融合蛋白抗菌肽Cecropin B-人溶菌酶奠定了基礎(chǔ)。 將重組后得到的克隆載體用引物擴增,凝膠電泳后回收目的條帶,然后將回收條帶和大腸桿菌表達載體pET32a用同樣的限制性內(nèi)切酶切割,用T4 DNA連接酶將兩者連接。經(jīng)測序及酶切鑒定融合基因Cecropin B-人溶菌酶已經(jīng)正確的連接到pET32a上。 將重組表達載體pET32a-CB-hLyso轉(zhuǎn)化至大腸桿菌BL21(DE3)和BL21(DE3)pLysS,然后經(jīng)IPTG誘導(dǎo)表達了融合蛋白抗菌肽Cecropin B-人溶菌酶并初步測定了活性。
[Abstract]:Antimicrobial peptide (anti-bacterial) is a kind of small molecular peptide produced by specific gene encoding in biological cells. It is an important molecular barrier for host to defend against the invasion of pathogenic microorganisms, and its formation and release is an integral part of the body's inflammatory response. Antimicrobial peptides not only have the ability of killing Gram-negative bacteria and Gram-positive bacteria, but also inhibit some viruses, fungi and protozoa, and have selective killing effect on some tumor cells. However, there were no obvious side effects on normal mammalian and insect cells. Human lysozyme (human lysozyme) has the ability to resist Gram-negative bacteria and some viruses. Human lysozyme is a kind of protein in human body and has natural compatibility with human body. In clinical application, it is safer than other lysozyme, without irritation and side effects. The aim of this study was to link Cecropin B and human lysozyme gene to the efficient expression vector of E. coli by gene recombination, and to further explore the antibacterial and antiviral activities of the fusion protein. In order to develop a higher activity of antibacterial and antiviral recombinant protein. In order to construct the gene cloning vectors of Cecropin B and human lysozyme (hLyso), the gene of antimicrobial peptide B was synthesized by overlapping region amplification method, the human lysozyme gene was removed from pUC118-hLvso, then fused into the clone vector according to the correct reading frame. The sequencing of the recombinant plasmid shows that the fusion gene Cecropin B-human Lysozyme has been correctly cloned into pBS-T vector, and the recombinant plasmid is transferred into E. coli so that the target gene can be preserved in E. coli and amplified in large quantities. It lays a foundation for the construction of expression vector of Cecropin B- human lysozyme. The recombinant clone vector was amplified by primers, and the target band was recovered by gel electrophoresis. Then, the recovered band and E. coli expression vector pET32a were digested with the same restriction endonuclease and ligated with T4 DNA ligase. The fusion gene Cecropin B- human lysozyme was correctly linked to pET32a by sequencing and restriction endonuclease digestion. The recombinant expression vector pET32a-CB-hLyso was transformed into Escherichia coli BL21 (DE3) and BL21 (DE3) pLysS. the fusion protein Cecropin B- human lysozyme was induced by IPTG and its activity was preliminarily determined.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

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