本文選題：家蠅 + 14-3-3ζ��； 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:從家蠅cDNA文庫(kù)中篩選獲得家蠅14-3-3ζ(MD14-3-3ζ)基因序列,對(duì)該序列及其編碼蛋白進(jìn)行分子特性分析,原核表達(dá),初步探討重組蛋白的抗菌活性及抗菌機(jī)制;檢測(cè)MD14-3-3ζ基因在家蠅生活史中各齡期及3齡幼蟲不同組織部位時(shí)空表達(dá)模式,初步探討經(jīng)大腸埃希菌誘導(dǎo)后,基因表達(dá)譜的變化,為進(jìn)一步探討其功能提供實(shí)驗(yàn)依據(jù)。方法:1、序列分析:采用生物信息學(xué)相關(guān)軟件,對(duì)家蠅14-3-3ζ基因及其編碼蛋白的基本理化性質(zhì)、信號(hào)肽、二級(jí)結(jié)構(gòu)等進(jìn)行分析,預(yù)測(cè)蛋白質(zhì)功能。2、cDNA克隆及原核表達(dá):根據(jù)MD14-3-3ζ的cDNA序列設(shè)計(jì)引物,PCR擴(kuò)增,構(gòu)建pET-28a(+)-MD14-3-3ζ重組質(zhì)粒,轉(zhuǎn)入大腸桿菌BL21(DE3)中,IPTG誘導(dǎo)表達(dá),SDS-PAGE分析表達(dá)產(chǎn)物的可溶性,通過(guò)鎳柱純化重組蛋白。3、Western blot及質(zhì)譜鑒定:將純化的MD14-3-3ζ蛋白免疫新西蘭大白兔,獲取多克隆抗體,Western blot鑒定重組蛋白;切下純化蛋白的SDS-PAGE條帶,送公司進(jìn)行質(zhì)譜分析鑒定。4、重組MD14-3-3ζ蛋白的抗菌活性檢測(cè)及抗菌功能初探:采用微量液體稀釋法,以大腸埃希菌、金黃色葡萄球菌為指示菌,檢測(cè)重組蛋白的抗菌活性;細(xì)胞內(nèi)膜滲透性實(shí)驗(yàn)檢測(cè)MD14-3-3ζ對(duì)大腸埃希菌細(xì)胞膜通透性的影響。5、MD14-3-3ζ基因時(shí)空表達(dá)模式的研究:1)收集家蠅生活史各發(fā)育階段(卵、各齡期、蛹、成蟲)的蟲體及3齡幼蟲不同組織(體壁、脂肪體、馬氏管、中腸、唾液腺、氣管),分別提取總RNA后并逆轉(zhuǎn)錄為cDNA,以家蠅RPS18為內(nèi)參基因,對(duì)不同樣本的MD14-3-3ζ基因表達(dá)情況進(jìn)行實(shí)時(shí)熒光定量PCR(qPCR)檢測(cè),所有實(shí)驗(yàn)進(jìn)行3個(gè)生物重復(fù),每個(gè)樣本重復(fù)實(shí)驗(yàn)3次。2)大腸埃希菌誘導(dǎo)后MD14-3-3ζ基因時(shí)空表達(dá)譜的變化:采用顯微注射的方法將細(xì)菌注入家蠅2齡晚期幼蟲,分別收集誘導(dǎo)后3 h、6 h、12 h、24 h、36 h、48 h蟲體,qPCR檢測(cè)不同時(shí)間點(diǎn)MD14-3-3ζ基因表達(dá)水平的變化,以注射PBS組為對(duì)照。結(jié)果:1、MD14-3-3ζ基因ORF全長(zhǎng)771 bp,編碼257個(gè)氨基酸,理論分子量29.35 kD;等電點(diǎn)4.87,屬于親水性的酸性蛋白,含有多種酶的結(jié)合位點(diǎn),有14-3-3家族結(jié)構(gòu)域和活性位點(diǎn),定位于細(xì)胞核中,二級(jí)結(jié)構(gòu)以α螺旋和無(wú)規(guī)則卷曲為主。2、成功構(gòu)建MD14-3-3ζ原核表達(dá)載體,IPTG誘導(dǎo)后,上清可溶表達(dá)重組蛋白,經(jīng)鎳柱純化,獲得純化的MD14-3-3ζ重組蛋白。3、Western blot結(jié)果顯示,得到與預(yù)計(jì)條帶大小相符的清晰條帶;質(zhì)譜分析顯示,重組蛋白序列與MD14-3-3ζ序列一致。4、體外抗菌試驗(yàn)結(jié)果:MD14-3-3ζ蛋白對(duì)大腸埃希菌、金黃色葡萄球菌均有抑制作用,MIC(minimal inhibitory concentration)值分別為0.16 mg/mL、0.25mg/mL。細(xì)胞內(nèi)膜滲透性實(shí)驗(yàn)顯示,隨著MD14-3-3ζ作用時(shí)間的延長(zhǎng),A430nm值從0.1左右上升至0.7,隨著時(shí)間的延長(zhǎng)而增加。5、時(shí)空表達(dá)模式研究:1)在不同發(fā)育時(shí)期,以卵期為參照,該基因在成蟲中表達(dá)量最高,表達(dá)量為成蟲2齡幼蟲蛹1齡幼蟲3齡幼蟲卵,成蟲比卵期上調(diào)了141.773倍(P0.05),2齡幼蟲則上調(diào)了79.7967倍(P0.05);在3齡幼蟲不同組織中,該基因在體壁表達(dá)量最高,其次為氣管(P0.05)和唾液腺(P0.01)。2)注射感染大腸埃希菌后,以PBS組為對(duì)照進(jìn)行計(jì)算,表明注射感染大腸埃希菌后3 h,MD14-3-3ζ基因出現(xiàn)明顯的上調(diào),上調(diào)了14.704倍(P0.05),其次為24 h,上調(diào)了2.503倍。36 h、48 h為下調(diào)趨勢(shì)(P0.01)。結(jié)論:1、本研究對(duì)家蠅14-3-3ζ基因進(jìn)行了分子特性分析,體外克隆、表達(dá),獲得并鑒定了MD14-3-3ζ重組蛋白;該重組蛋白在體外對(duì)金黃色葡萄球菌、大腸埃希菌均有抗菌效果,對(duì)大腸埃希菌抗菌活性較好;并能改變其細(xì)胞內(nèi)膜的通透性。2、MD14-3-3ζ基因在家蠅不同生長(zhǎng)時(shí)期及3齡幼蟲不同組織中均有表達(dá),經(jīng)大腸埃希菌誘導(dǎo)后,該基因的表達(dá)量在3 h出現(xiàn)了明顯的上調(diào),提示該基因參與了家蠅的免疫防御過(guò)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.

【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R384.2

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