抗菌肽Cecropin B-人溶菌酶融合蛋白基因的设计、克隆及在大肠杆菌中的表达

发布时间：2018-06-27 12:56

本文选题：抗菌肽 + 天蚕素B　；参考：《西北大学》2005年硕士论文

【摘要】：抗菌肽(anti-bacterial peptides,简称ABP)是生物细胞特定基因编码产生的一类小分子多肽,是宿主防御病原微生物入侵的重要分子屏障,其生成和释放是机体炎症反应的组成部分。抗菌肽不仅对革兰氏阴性细菌和革兰氏阳性细菌具有高效广谱的杀灭能力,而且对某些病毒、真菌和原虫也具有抑杀作用;同时它对一些肿瘤细胞具有选择性杀伤作用,但对正常哺乳动物和昆虫细胞却无明显的毒副作用。人溶菌酶(human lysozyme)具有抗革兰氏阴性细菌和抗一些病毒的能力。人溶菌酶是人体内的一种蛋白质,和人体具有天然相容性。在临床上应用时,比其它溶菌酶更安全,没有刺激性和副作用。本实验目的是想通过基因重组,以融合的形式将Cecropin B和人溶菌酶基因连接至高效的大肠杆菌表达载体上并进行表达,进一步探讨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组蛋白。为了构建抗菌肽B(Cecropin B)和人溶菌酶(hLyso)的基因克隆载体,通过重叠区扩增法人工合成抗菌肽B基因,从pUC118-hLvso上卸下人溶菌酶基因,然后按照正确的阅读框架融合并重组至克隆载体中。通过对重组质粒的测序,表明融合基因Cecropin B-human Lysozyme已经正确克隆至pBS-T载体中,将重组质粒转入大肠杆菌里,使目的基因能够在大肠杆菌里保存并且大量扩增,为构建表达载体表达融合蛋白抗菌肽Cecropin B-人溶菌酶奠定了基础。将重组后得到的克隆载体用引物扩增,凝胶电泳后回收目的条带,然后将回收条带和大肠杆菌表达载体pET32a用同样的限制性内切酶切割,用T4 DNA连接酶将两者连接。经测序及酶切鉴定融合基因Cecropin B-人溶菌酶已经正确的连接到pET32a上。将重组表达载体pET32a-CB-hLyso转化至大肠杆菌BL21(DE3)和BL21(DE3)pLysS,然后经IPTG诱导表达了融合蛋白抗菌肽Cecropin B-人溶菌酶并初步测定了活性。
[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.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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