马尔尼菲青霉Atf21和Phx1基因功能与角质酶基因原核表达研究

发布时间：2018-06-02 23:28

本文选题：马尔尼菲青霉菌 + 基因敲除　；参考：《广西医科大学》2017年硕士论文

【摘要】：马尔尼菲青霉真菌(Penicillium marneffei PM)是青霉真菌属中唯一的一种温度依赖性的双相型条件致病真菌,它是马尔尼菲青霉真菌病(Penicilliosis marneffei PSM)的病原菌,25℃时以菌丝相生长,在37℃体外培养或者感染人体后转换为致病性酵母相生长;主要流行区域在东南亚地区,PM对免疫功能低下的患者感染性强,对感染者造成致命性的全身性感染。目前,马尔尼菲青霉真菌双相转换和致病性的分子机制尚不清楚。细胞转录因子是细胞感受外界信号后调控基因表达的关键分子,许多转录因子在调控基因表达的同时其基因自身的表达会受到调控。深入研究转录因子及其调控基因功能探讨马尔尼菲青霉双相转换与致病性分子机制,可为马尔尼菲青霉病防治和药物开发提供科学依据[1-6]。目的:1、本课题组前期进行的马尔尼菲青霉菌链特异性转录组的测序分析发现,编码含亮氨酸拉链(bZIP)的转录因子基因Atf21和编码含有同源异型盒(homeobox)结构域的转录因子基因Phx1在双相转换过程中差异表达显著,本课题通过构建马尔尼菲青霉菌Atf21和Phx1基因缺失突变体,对转录因子基因Atf21和Phx1的功能进行了初步研究,探讨马尔尼菲青霉双相转换与致病性分子机制,可为马尔尼菲青霉病防治和药物开发提供科学依据。2、本课题组前期研究发现编码角质酶转录因子β亚基基因(Ctf1β)及其调控的角质酶基因在酵母相表达显著升高。本课题拟通过克隆马尔尼菲青霉菌角质酶的基因,构建角质酶原核表达重组菌,进行角质酶的活性鉴定与分析,获得重组蛋白为进一步制备马尔尼菲青霉菌角质酶抗体和探讨该抗体在防治马尔尼菲青霉病中的应用奠定基础。方法:1、用实时荧光定量方法分析马尔尼菲青霉菌株gxff在双相转换过程中基因表达量和基因表达特征。2、利用同源重组方法,构建目的基因敲除突变株,然后与spm4野生菌株进行对比研究,初步研究和分析突变缺失基因的功能。3、通过引物设计、pcr扩增马尔尼菲青霉菌角质酶基因序列,构建诱导型表达载体,并对重组质粒进行pcr鉴定和质粒的测序;用碱式滴定法测定酶活性;提取蛋白对角质酶进行表达鉴定。结果:1、atf21和phx1基因在马尔尼菲青霉菌双相转换不同时相的表达进行分析结果表明,在马尔尼菲青霉菌株gxff中atf21和phx1基因表达在菌丝相生长状态显著高于在酵母相生长状态,与在马尔尼菲青霉菌株frr2161中转录组测序分析atf21基因表达结果一致;atf21在双相转换早期基因表达明显改变,在双向转化过程中phx1基因表达出现明显改变时间较晚。2、成功构建了atf21和phx1基因敲除菌株,与对照菌spm4对比,atf21突变菌株对cu更加敏感,phx1突变菌株对cu的敏感性无影响。3、成功克隆了马尔尼菲青霉菌角质酶基因,构建了诱导型表达载体并进行测序验证;构建了马尔尼菲青霉菌角质酶基因大肠杆菌原核表达重组菌株;用碱式滴定法测定重组菌株酶活性约为对照菌1.7倍,粗酶液的酶活性可达21.8u/ml。结论:1、马尔尼菲青霉菌atf21和phx1基因在双相转换不同时相表达差异显著,atf21和phx1基因表达在菌丝相生长状态显著高于在酵母相生长状态;atf21在双相转换早期基因表达明显改变,在双向转化过程中phx1基因表达出现明显改变时间较晚。2、成功构建了atf21和phx1基因敲除菌株,创新性地发现马尔尼菲青霉菌转录因子基因atf21具有调控铜离子代谢功能,为深入探讨马尔尼菲青霉双相转换与致病性分子机制奠定了基础。3、成功克隆了马尔尼菲青霉菌角质酶基因,构建了马尔尼菲青霉菌角质酶基因大肠杆菌原核表达重组菌株,为进一步获得重组蛋白制备马尔尼菲青霉菌角质酶抗体和探讨该抗体在防治马尔尼菲青霉病中的应用奠定基础。
[Abstract]:Penicillium marneffei PM is the only temperature dependent biphasic pathogenic fungus in the genus Penicillium. It is the pathogen of Penicilliosis marneffei PSM (Penicilliosis marneffei PSM). At 25 C, it is grown in mycelium and converted to pathogenic yeast at 37 C in vitro or infected with human body. The main epidemic area is in Southeast Asia, PM is highly infectious to patients with low immune function and causes fatal systemic infection to infected people. At present, the molecular mechanism of biphasic conversion and pathogenicity of Penicillium marneffy fungi is not clear. On the other hand, many transcriptional factors will be regulated while regulating gene expression. Further study on the function of transcription factors and their regulatory genes to study the biphasic transformation and pathogenicity of Penicillium marneffei can provide scientific basis for the prevention and control of Penicillium marneffei and the development of drug [1-6].. 1. The sequence analysis of Penicillium marneffei chain specific transcriptome found that the transcriptional factor gene Atf21 containing leucine zipper (bZIP) and the transcriptional factor gene Phx1 containing the homologous type box (homeobox) domain were expressed differently in the biphasic transformation process. The subject was constructed by the construction of Penicillium marneffei Atf21 and the Atf21 of Penicillium marneffei. The function of Phx1 gene deletion mutant and the function of the transcription factor gene Atf21 and Phx1 were preliminarily studied. The biphasic conversion and pathogenic molecular mechanism of Penicillium marneffei could provide a scientific basis for the prevention and control of Penicillium marneffei and the development of drug,.2. In the previous study, the keratinocyte transcription factor beta subunit gene (Ctf1 beta) was developed. The expression of the keratinase gene and its regulated genes in the yeast phase increased significantly. This topic is to clone the keratinase gene of Penicillium marneffy, construct the recombinant bacteria of the keratinocyte, identify and analyze the activity of the keratinase, and obtain the recombinant protein for the further preparation of the antibody to the Penicillium marnffy keratinase and to discuss the antibody against this antibody. The basis for the application of the treatment of Penicillium marneffei was established. Method: 1, the gene expression and gene expression of Penicillium marneffei strain gxff were analyzed by real time fluorescence quantitative method (.2). The target gene knockout mutant was constructed by homologous recombination method, and then compared with the wild strain of spm4, the preliminary study was carried out. And analyze the function.3 of the mutant deletion gene. Through the primer design, PCR amplification of the Penicillium marneffy keratinase gene sequence, the inducible expression vector was constructed, the recombinant plasmid was identified by PCR and the plasmid was sequenced. The enzyme activity was determined by the alkaline titration, and the expression of the protein diagonal enzyme was identified. The results were as follows: 1, atf21 and phx1 genes were found. The results showed that the expression of atf21 and phx1 gene expression in the mycelial phase of Penicillium marneffei gxff was significantly higher than that in the yeast phase, which was consistent with the atf21 gene expression results in the transcriptional sequence analysis in Penicillium marneffei frr2161; atf21 was in the form of atf21 gene expression. The expression of gene expression in the early stage of biphasic transformation was obviously changed. In the process of bi-directional transformation, the expression of phx1 gene was obviously changed later.2. The atf21 and phx1 gene knockout strains were successfully constructed. Compared with the control bacteria spm4, the atf21 mutant strain was more sensitive to Cu, and the sensitivity of phx1 mutant to Cu was not affected.3, and the Penicillium marneffei was cloned successfully. The inducible expression vector was constructed and sequenced, and the recombinant strain of Escherichia coli was constructed. The enzyme activity of the recombinant strain was about 1.7 times that of the control bacteria by alkaline titration, and the enzyme activity of the crude enzyme could reach 21.8u/ml. conclusion: 1, Penicillium marneffei atf21 and phx1 The expression of atf21 and phx1 gene expression in the mycelial phase was significantly higher than that in the yeast phase, and the expression of atf21 in the early stage of biphasic transformation was significantly changed, and the expression of phx1 gene in the process of biphasic transformation was significantly changed during the bi-directional transformation, and the atf21 and phx1 gene knockout were successfully constructed. It has been found that the Penicillium marneffei transcriptional factor gene atf21 has the function of regulating copper ion metabolism, which lays a foundation for the in-depth study of the biphasic transformation and pathogenic molecular mechanism of Penicillium marneffei, which has successfully cloned the Penicillium marneffei keratinase gene and constructed the coliform pole of the Penicillium marneffei keratinase gene. The recombinant strain expressed the recombinant strain to prepare the recombinant protein of Penicillium marneffei and explore the application of the antibody in the prevention and control of Penicillium marneffei.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379

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