家蝇14-3-3ζ基因的表达及抗菌功能初探

发布时间：2018-05-13 19:45

本文选题：家蝇 + 14-3-3ζ　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:从家蝇cDNA文库中筛选获得家蝇14-3-3ζ(MD14-3-3ζ)基因序列,对该序列及其编码蛋白进行分子特性分析,原核表达,初步探讨重组蛋白的抗菌活性及抗菌机制;检测MD14-3-3ζ基因在家蝇生活史中各龄期及3龄幼虫不同组织部位时空表达模式,初步探讨经大肠埃希菌诱导后,基因表达谱的变化,为进一步探讨其功能提供实验依据。方法:1、序列分析:采用生物信息学相关软件,对家蝇14-3-3ζ基因及其编码蛋白的基本理化性质、信号肽、二级结构等进行分析,预测蛋白质功能。2、cDNA克隆及原核表达:根据MD14-3-3ζ的cDNA序列设计引物,PCR扩增,构建pET-28a(+)-MD14-3-3ζ重组质粒,转入大肠杆菌BL21(DE3)中,IPTG诱导表达,SDS-PAGE分析表达产物的可溶性,通过镍柱纯化重组蛋白。3、Western blot及质谱鉴定:将纯化的MD14-3-3ζ蛋白免疫新西兰大白兔,获取多克隆抗体,Western blot鉴定重组蛋白;切下纯化蛋白的SDS-PAGE条带,送公司进行质谱分析鉴定。4、重组MD14-3-3ζ蛋白的抗菌活性检测及抗菌功能初探:采用微量液体稀释法,以大肠埃希菌、金黄色葡萄球菌为指示菌,检测重组蛋白的抗菌活性;细胞内膜渗透性实验检测MD14-3-3ζ对大肠埃希菌细胞膜通透性的影响。5、MD14-3-3ζ基因时空表达模式的研究:1)收集家蝇生活史各发育阶段(卵、各龄期、蛹、成虫)的虫体及3龄幼虫不同组织(体壁、脂肪体、马氏管、中肠、唾液腺、气管),分别提取总RNA后并逆转录为cDNA,以家蝇RPS18为内参基因,对不同样本的MD14-3-3ζ基因表达情况进行实时荧光定量PCR(qPCR)检测,所有实验进行3个生物重复,每个样本重复实验3次。2)大肠埃希菌诱导后MD14-3-3ζ基因时空表达谱的变化:采用显微注射的方法将细菌注入家蝇2龄晚期幼虫,分别收集诱导后3 h、6 h、12 h、24 h、36 h、48 h虫体,qPCR检测不同时间点MD14-3-3ζ基因表达水平的变化,以注射PBS组为对照。结果:1、MD14-3-3ζ基因ORF全长771 bp,编码257个氨基酸,理论分子量29.35 kD;等电点4.87,属于亲水性的酸性蛋白,含有多种酶的结合位点,有14-3-3家族结构域和活性位点,定位于细胞核中,二级结构以α螺旋和无规则卷曲为主。2、成功构建MD14-3-3ζ原核表达载体,IPTG诱导后,上清可溶表达重组蛋白,经镍柱纯化,获得纯化的MD14-3-3ζ重组蛋白。3、Western blot结果显示,得到与预计条带大小相符的清晰条带;质谱分析显示,重组蛋白序列与MD14-3-3ζ序列一致。4、体外抗菌试验结果:MD14-3-3ζ蛋白对大肠埃希菌、金黄色葡萄球菌均有抑制作用,MIC(minimal inhibitory concentration)值分别为0.16 mg/mL、0.25mg/mL。细胞内膜渗透性实验显示,随着MD14-3-3ζ作用时间的延长,A430nm值从0.1左右上升至0.7,随着时间的延长而增加。5、时空表达模式研究:1)在不同发育时期,以卵期为参照,该基因在成虫中表达量最高,表达量为成虫2龄幼虫蛹1龄幼虫3龄幼虫卵,成虫比卵期上调了141.773倍(P0.05),2龄幼虫则上调了79.7967倍(P0.05);在3龄幼虫不同组织中,该基因在体壁表达量最高,其次为气管(P0.05)和唾液腺(P0.01)。2)注射感染大肠埃希菌后,以PBS组为对照进行计算,表明注射感染大肠埃希菌后3 h,MD14-3-3ζ基因出现明显的上调,上调了14.704倍(P0.05),其次为24 h,上调了2.503倍。36 h、48 h为下调趋势(P0.01)。结论:1、本研究对家蝇14-3-3ζ基因进行了分子特性分析,体外克隆、表达,获得并鉴定了MD14-3-3ζ重组蛋白;该重组蛋白在体外对金黄色葡萄球菌、大肠埃希菌均有抗菌效果,对大肠埃希菌抗菌活性较好;并能改变其细胞内膜的通透性。2、MD14-3-3ζ基因在家蝇不同生长时期及3龄幼虫不同组织中均有表达,经大肠埃希菌诱导后,该基因的表达量在3 h出现了明显的上调,提示该基因参与了家蝇的免疫防御过程。
[Abstract]:Objective: to select the 14-3-3 zeta (MD14-3-3 zeta) gene sequence of housefly from the cDNA Library of housefly, to analyze the molecular characteristics of the sequence and its encoded protein, to express the prokaryotic expression, to investigate the antibacterial activity and the antibacterial mechanism of the recombinant protein, and to detect the temporal and spatial expression of the MD14-3-3 zeta gene in the different tissues of the 3 instar larvae and the age of the housefly. Model, preliminary study on the changes of gene expression profiles after the induction of Escherichia coli and provide experimental basis for further exploring its function. Methods: 1, sequence analysis: the basic physicochemical properties of 14-3-3 zeta gene and its encoded protein, signal peptide and two grade structure were analyzed by bioinformatics related software, and the function of protein was predicted, and the function of protein was predicted, and the function of.2 was predicted. CDNA cloning and prokaryotic expression: Based on the cDNA sequence of MD14-3-3 zeta, primers were designed, PCR amplification, construction of pET-28a (+) -MD14-3-3 zeta recombinant plasmid, into Escherichia coli BL21 (DE3), IPTG induced expression, SDS-PAGE analysis of the soluble expression products, purified recombinant egg white.3 by nickel column, Western enrichment and mass spectrometry identification: the purified zeta protein exempts The New Zealand white rabbit, obtained polyclonal antibody, Western blot identification of recombinant protein, cut down the SDS-PAGE band of purified protein, and sent the company to identify.4 by mass spectrometry analysis, detection of the antibacterial activity of recombinant MD14-3-3 zeta protein and its antibacterial function: using microdilution method, Escherichia coli and Staphylococcus aureus as indicator bacteria, and detection of Escherichia coli and Staphylococcus aureus The antibacterial activity of recombinant protein; the influence of MD14-3-3 zeta on the membrane permeability of Escherichia coli.5, MD14-3-3 zeta gene spatio-temporal expression pattern: 1) the insect body of the life history of the housefly (egg, age, pupa, adult) and the different tissues of the 3 instar larvae (body wall, fat body, martensitic tube, midgut, spittle) Liquid gland, trachea), after extracting total RNA and reverse transcriptase cDNA, using housefly RPS18 as internal reference gene, real-time fluorescence quantitative PCR (qPCR) detection of MD14-3-3 zeta gene expression in different samples, 3 Biological repetitions in all experiments, 3.2 in each experiment, and time and space expression profiles of MD14-3-3 zeta gene induced by Escherichia coli induced by Escherichia coli. Microinjection was used to inject bacteria into the late 2 instar larvae of Musca domestica to collect 3 h, 6 h, 12 h, 24 h, 36 h, 48 h body after induction. QPCR detected the changes in the expression level of MD14-3-3 zeta gene at different time points. The results were as follows: 1, MD14-3-3 Zeta gene ORF 771 BP, 257 amino acids, 29.35 theoretical molecular weights; The isoelectric point 4.87, which belongs to the hydrophilic acid protein, contains the binding site of a variety of enzymes, has the 14-3-3 family domain and the active site, is located in the nucleus. The two stage structure is mainly.2 with alpha helix and irregular curl, and the MD14-3-3 zeta prokaryotic expression vector is successfully constructed. After the induction of IPTG, the supernatant can dissolve the recombinant protein and purified by nickel column and obtained pure. The MD14-3-3 zeta recombinant protein.3, Western blot results showed that a clear strip coincide with the predicted band size; mass spectrometry analysis showed that the recombinant protein sequence was consistent with the MD14-3-3 zeta sequence.4, and in vitro antibacterial test results: the MD14-3-3 zeta protein had inhibitory effect on Escherichia coli and Staphylococcus aureus, MIC (minimal inhibitory concen). Tration) values were 0.16 mg/mL respectively. The permeability test of 0.25mg/mL. cell intima showed that the A430nm value increased from about 0.1 to 0.7 with the prolongation of the time of MD14-3-3 zeta. The time and space expression pattern was increased, and the spatio-temporal expression pattern was studied: 1) at different developmental stages, the expression of the gene was the highest in the adult. The eggs of 3 instar larvae of 1 instar larvae of 2 instar larvae were up to 141.773 times higher than the egg period (P0.05), and the 2 instar larvae increased by 79.7967 times (P0.05). In the different tissues of the 3 instar larvae, the expression of the gene was the highest in the body wall, followed by the infection of the trachea (P0.05) and the salivary gland (P0.01).2), and the PBS group was used as the control. After 3 h infection of Escherichia coli, the MD14-3-3 zeta gene increased obviously, up 14.704 times (P0.05), followed by 24 h, up 2.503 times.36 h, and 48 h as a downward trend (P0.01). Conclusion: 1, the molecular characterization of the 14-3-3 zeta gene in the housefly was analyzed, the expression was cloned in vitro, and the MD14-3-3 zeta recombinant protein was obtained and identified. The recombinant protein has antibacterial effect on Staphylococcus aureus and Escherichia coli in vitro, and has good antibacterial activity against Escherichia coli, and can change the permeability of the endometrium.2. The MD14-3-3 zeta gene is expressed in different growth period and 3 instar larvae of housefly, and the expression of this gene is 3 h after the induction of Escherichia coli. A significant up-regulated expression suggested that the gene was involved in the immune defense process of housefly.

【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R384.2

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