【作者】 陈宝玉；

【导师】 邓旭明；

【作者基本信息】 吉林大学 ， 基础兽医学， 2006， 硕士

【摘要】 哺乳动物防御素是近年来发现的广泛存在于哺乳动物体内的一类阳离子抗菌肽,不仅具有广谱抗微生物作用,而且还能杀伤多种肿瘤细胞,其潜在药用价值巨大。本研究利用基因工程原理及现代分子生物学实验技术,通过反转录克隆获得鼠小肠隐窝防御素cryptdin 4(以下简写为:crp4)基因,以便对crp4的结构和功能进行研究。根据GenBank中报道的crp4基因的序列,为了得到大量的防御素crp4,我们利用大肠杆菌表达系统和杆状病毒表达系统表达crp4基因,将克隆得到的crp4成熟肽片段转到原核载体pMAL-p2x、pET-28a(+)和杆状病毒表达载体pFastBac1TM中,得到重组表达质粒pMAL-129、pET-129和pFast-129。经SDS-PAGE电泳检测后,结果显示:几个重组表达质粒在各自的宿主菌和细胞中均得到成功表达,体外抑菌实验结果显示得到的目的蛋白对不同的指示菌均有不同程度的抑菌效果。更多还原

【Abstract】 The defensins of paneth cell is a natural antibiotic peptide,which is secreted fromthe intestine crypts in mammalian.It is very fast about synthesis and secrete velocity insmall intestine.There have broad-spectrum and high-performance antimicrobialactivity,which conduce to enteric host immunization,and which make defensins to payclose attention in enteric innate immunity.Furthermore,it is thought highly how torestrain bacterium aversion and prevent enterogenic infection.Therefore,it is importantsignificance to profound research the defensins and application in clinic.Mouse crp4 is a kind of defensin,which secreted by paneth cell of mouse ,itsterms cryptdins.crp4 is endogenous antibiosis product of phagocytic cell andenterocyte.It effected microenvironment of extracellular together with lysozyme,secretion type phosphatidolipase A2, antimicrobial peptide,which had been secred byintestine. Paneth cells at the base of the crypts of Lieberkuhn in the small intestinesecrete apically oriented granules as components of innate immunity.According cryptdin4 gene order publicated in GenBank, we designed and synthetized three specificprimers,then to detect the mRNA level of cryptdin 4 in different daytimes and differentsegement of small intestine in 129/SVJ mouse by quantitation real-time PCR,and thenwe found that older is the mouse,higher is the mRNA level of the cryptdin4 expression,and which had a highest level in ileum.So we chosed the terminal ileum mRNA of129/SVJ and Kunming mouse about 30 days to undertake the subsequentresearch.Through used RT-PCR to amplify and clone crp41 gene, which is thestructural gene of cryptdin 4.After sequence analysis,the result displayed that crp41gene contains 279 nucleotides which encode 93 amino acides,in which have 59 aminoacides encode signal peptide and precursor of N-terminal region, another 34 residesencode the mature peptide of cryptdin 4 in C-terminal region. By RT-PCR to amplifyagain,We cloned crp42 gene which is the mature peptide of cryptdin 4.The result ofidentification displayed crp42 gene whose length is 102 resides had obtainedsuccessful by PCR and enzyme digestion,and we found their orders are coincidence withcrp42 of 129/SVJ and its publicated in GenBank,but when compared among crp42who come from different mouse strains,which have quite discrepancy. Sequencealignment showed that the maximal homology of crp42 genes is 91.8%.But if we want to undertake elementary research and exploratory developmentbetter,it is imperative to get sufficient cryptdin 4 . So three heterologous E.coli expres-sion systems and one Baculovirus expression system were used to produce recombi-nation crp42. The DNA fragment crp42 was cloned into vectors pMAL-p2x, pET-28aand pFastBac1TM respectively. The four constructed expression vectors, namely MBP-129, His-tag-129 and pFast-129 ,respectively, were transformed into E.coli host strainsand insects cell sf9. SDS-PAGE analysis of protein prepa -rations indicated high levelexpression of four fusion proteins, and MBP-129,His-tag-129 and pFast-129 wereshown to be soluble.Inhibitory activity of fusion proteins were detected by agar diffu-sion test.The pFast-129 protein displays evident activity against Staphylococcus aureus,Salmonella typhi,etc,and His-tag-129 and MBP-129 showed visible activity against Sta-phylococcus aureus.After purification by affinity chromatograph to His-tag-129 andMBP-129,we found their result was poor.Because active protein can be obtained through the DNA fragment crp42 expressedin pMAL-p2x and pFastBac1,we will optimize their conditions for better expression infuture,which have important directions to approach how to boost the activity of cryptdin4 and to design and synthetize another defensins reasonable,which may provide theoryevidences for the distribution of cryptdin 4 in intestinal tract and the research of tran-genic plant.更多还原