本文選題：非洲鴕鳥 + B淋巴細胞刺激因子　； 參考：《華中農(nóng)業(yè)大學》2016年博士論文

【摘要】：B細胞刺激因子(B cell activating factor,BAFF)是腫瘤壞死因子(tumor necrosis factor,TNF)家族中的主要成員之一,是具有維持B細胞功能穩(wěn)定作用的重要細胞因子。BAFF與T細胞的活化、B淋巴細胞的發(fā)育及體液免疫等直接相關(guān)。BAFF可以使尚未完全成熟的B細胞分化成更加成熟的B細胞,同時BAFF還可以延長B細胞的存活、促進抗體的產(chǎn)生,BAFF因其在B細胞發(fā)育及自身免疫病中起非常重要的作用而日益受人關(guān)注和重視。非洲鴕鳥因其生長速度快,適應(yīng)性廣,飼料轉(zhuǎn)化率高,近年來其養(yǎng)殖規(guī)模穩(wěn)步發(fā)展。但是在非洲鴕鳥的養(yǎng)殖過程中,3月齡以內(nèi)的雛鴕鳥對多種病原的易感性較強,非常容易發(fā)生各類疾病,給非洲鴕鳥養(yǎng)殖業(yè)造成巨大的經(jīng)濟損失。硼(Boron,B)是一種微量元素,廣泛分布于自然界。硼具有多種生物學作用。近年的研究表明,硼可能是某些動物生長發(fā)育和新陳代謝過程中不可或缺的微量元素。對動物的大量研究發(fā)現(xiàn)硼可以影響機體的免疫功能,生理劑量的硼具有止痛、抗炎和增強免疫等作用,較高劑量的硼具有毒性作用,可導致免疫器官發(fā)育受阻和免疫功能低下。本研究以非洲鴕鳥為對象,參考已有的鳥類BAFF基因序列設(shè)計引物,擴增鴕鳥BAFF基因,通過基因克隆、原核表達和熒光定量PCR等方法研究BAFF基因在各免疫器官內(nèi)的表達及其蛋白的生物學活性。同時通過在飲水中人工添加不同劑量的硼,利用實時熒光定量PCR等方法檢測分析硼對BAFF基因在非洲雛鴕鳥不同組織中表達情況的影響,為探究硼的營養(yǎng)作用和免疫毒性作用奠定基礎(chǔ),為非洲鴕鳥的不同病原免疫防御機制的研究提供有價值的參考,同時也可為非洲鴕鳥及其它鳥類的疾病防治提供科學的依據(jù)。主要研究內(nèi)容與結(jié)果如下:1、非洲鴕鳥BAFF基因的克隆與序列分析參考GenBank中公布的其它禽類BAFF的基因序列設(shè)計引物,以非洲鴕鳥法氏囊總RNA為模板,經(jīng)RT-PCR擴增,成功擴增出非洲鴕鳥BAFF基因全序列。所得序列用MEGA 5.05軟件和Signal P 4.1 server軟件對其堿基序列和氨基酸序列進行同源性分析和進化樹分析。結(jié)果表明:非洲鴕鳥BAFF基因全長867 bp,編碼288個氨基酸;同源性分析發(fā)現(xiàn),非洲鴕鳥BAFF基因氨基酸序列與雞BAFF基因氨基酸序列的同源性最高,達52%,其次是鵪鶉(qs BAFF,51%)、鴨(d BAFF,50%)、鵝(g BAFF,50%)和鴿子(do BAFF,49%);非洲鴕鳥BAFF基因堿基序列進化樹分析顯示非洲鴕鳥BAFF基因與雞、鵪鶉的同源性最近,其次是鴨、鵝和鴿子;氨基酸序列進化樹分析結(jié)果顯示分析所用的氨基酸序列分為三支,一支包含了分析所用的所有鳥類,一支包含了所用的魚類,另一支包含了所有的哺乳動物,非洲鴕鳥BAFF基因氨基酸序列與其它鳥類的同源性較近。這些結(jié)果可為鳥類BAFF基因的進化研究以及分子生物學研究提供參考。2、非洲鴕鳥BAFF基因的原核表達根據(jù)本研究克隆獲得的非洲鴕鳥BAFF基因序列設(shè)計特異性引物,分別在上、下游引物中加入Eco R I和Hind III酶切位點,成功擴增出非洲鴕鳥BAFF基因后,將該基因片段克隆至p ET-28a原核表達載體;PCR、酶切鑒定結(jié)果表明成功構(gòu)建了陽性重組表達質(zhì)粒p ET-Os BAFF。將該重組質(zhì)粒轉(zhuǎn)化入大腸桿菌Rosetta(DE3)中,經(jīng)IPTG誘導表達,將表達產(chǎn)物進行SDS-PAGE電泳分析、Western blotting驗證,最終確定該重組蛋白大小為32.2 k D。3、非洲鴕鳥BAFF基因的組織表達研究采用real-time PCR方法分析了非洲鴕鳥BAFF基因在18種組織中的m RNA表達情況。結(jié)果顯示:BAFF廣泛分布于所檢測的組織中,但在不同組織中的表達量有差異。非洲鴕鳥BAFF在法氏囊、胸腺、脾臟和骨髓等組織中表達量較高,其次是肝、腎臟、氣管、肺、胃和腸,在其它檢測的組織中的表達量相對較低,最低的是心臟和腦,幾乎無表達。非洲鴕鳥BAFF基因組織分布的差異提示其在不同組織中所發(fā)揮的主要功能可能有所不同。4、非洲鴕鳥BAFF基因重組蛋白的生物學活性研究哺乳動物和其它鳥類的BAFF是B細胞存活的重要影響因子。為驗證非洲鴕鳥的BAFF蛋白是否也可以延長B細胞的存活時間。對表達獲得的非洲鴕鳥BAFF基因重組蛋白進行純化,利用MTT實驗法測定該重組蛋白在非洲鴕鳥B淋巴細胞增殖中的作用。結(jié)果顯示:添加了純化的重組蛋白或添加純化的重組蛋白和PMA的試驗組的B淋巴細胞培養(yǎng)24h時,其細胞生長情況與重組蛋白的添加濃度有明顯的相關(guān)性,當添加12μg/m L的重組蛋白或者16μg/m L的重組蛋白和2μg/m L的PMA時,B淋巴細胞生長密度達到最大值,而對照組添加PBS及BSA組,其B淋巴細胞均無明顯增多。對小鼠淋巴細胞增殖的影響試驗表明,非洲鴕鳥BAFF基因重組蛋白在體外可以產(chǎn)生與非洲鴕鳥法氏囊B淋巴細胞增殖試驗類似的結(jié)果,同樣可以刺激小鼠B淋巴細胞存活/增殖。LPS刺激實驗結(jié)果表明,非洲鴕鳥BAFF基因m RNA表達水平在法氏囊淋巴細胞受LPS刺激后的6、12、24及48小時后均高于對照組。非洲鴕鳥BAFF基因m RNA表達水平自LPS刺激后6 h開始大幅上調(diào),最大值出現(xiàn)在刺激后24 h;與對照組相比,直到刺激后48 h其m RNA表達一直保持在相對較高水平(p0.01)。5、飲水硼對非洲鴕鳥BAFF基因組織表達的影響為探究微量元素硼對非洲鴕鳥BAFF基因組織表達的影響,取健康1日齡非洲鴕鳥48羽,將其隨機分為6組,每組8羽。分別在飲水中添加0 mg/L(對照組),40 mg/L,80 mg/L,160 mg/L,320 mg/L,640 mg/L劑量的硼酸,分別于1d,45d和90d取材,分別取法氏囊、胸腺、骨髓、脾等組織,提取總RNA作為模板,采用熒光定量PCR法分析不同飲水硼組非洲鴕鳥BAFF基因的組織表達情況。結(jié)果發(fā)現(xiàn):非洲鴕鳥BAFF基因m RNA的組織表達與飲水中硼的添加劑量相關(guān)。低劑量時,所測8種組織中BAFF基因m RNA的表達隨飲水中硼添加劑量的增加而升高;當硼添加劑量到一定程度時,隨著添加劑量的增大,BAFF基因的表達反而降低。45日齡時,骨髓、脾、胸腺、肺等4種組織中BAFF基因表達的最大值為160 mg/L試驗組,法氏囊、肝、腎等3種組織中BAFF基因表達的最大值為80 mg/L試驗組,均顯著高于對照組(P0.05);胃組織中BAFF基因表達的最大值為80 mg/L試驗組,但與對照組相比差異性不顯著。90日齡時,骨髓、法氏囊、肝、脾、胸腺、腎等6種組織中BAFF基因表達的最大值為80 mg/L試驗組,肺組織中BAFF基因表達的最大值為160 mg/L試驗組,均顯著高于對照組(P0.05);胃組織中BAFF基因表達的最大值為160 mg/L試驗組,但與對照組相比無顯著性差異。
[Abstract]:The B cell stimulating factor (B cell activating factor, BAFF) is one of the main members of the tumor necrosis factor (tumor necrosis factor, TNF) family. It is an important cytokine with the function of maintaining the functional stability of B cells. B cells differentiate into more mature B cells, and BAFF can also prolong the survival of B cells and promote the production of antibodies. BAFF is becoming more and more important because of its very important role in the development of B cell and autoimmune disease. The African ostrich is fast growing, well adapted, high feed conversion rate and its breeding scale in recent years. Steady development. But in the farming process of African ostrich, the ostrich within 3 month old is more susceptible to a variety of pathogens, it is very easy to occur various diseases and cause huge economic losses to the African ostrich aquaculture industry. Boron (Boron, B) is a kind of trace element, widely distributed in nature. Boron has many biological functions. Boron may be an indispensable trace element in the growth and metabolism of some animals. A large number of studies on animals have found that boron can affect the immune function of the body. Boron in the physiological dose has the effects of analgesic, anti-inflammatory and enhanced immunity. The high dose of boron has the toxic effect, which can cause the development of immune organs to be blocked. In this study, we used the African ostrich as the object, designed primers to amplify the ostrich BAFF gene by using the BAFF gene sequence of the existing birds, and studied the expression of the BAFF gene in the immune organs and the biological activity of the protein by gene cloning, prokaryotic expression and fluorescence quantitative PCR. The effects of Boron on the expression of BAFF gene in different tissues of African ostrich were detected by real time fluorescence quantitative PCR, which lay the foundation for exploring the nutritional and immune toxicity of boron, and provided valuable reference for the study of different pathogenic immune defense mechanisms of African ostrich. The main research contents and results are as follows: 1, the cloning and sequence analysis of the BAFF gene of African ostrich, reference to the sequence design primers of other avian BAFF published in GenBank, using the total RNA of the African ostrich bursa as the template, the African ostrich B was amplified by RT-PCR, and the African ostrich B was amplified successfully. The sequence of the AFF gene was analyzed by MEGA 5.05 software and Signal P 4.1 server software. The results showed that the total length of the African ostrich BAFF gene was 867 BP and encoded 288 amino acids, and the homology analysis showed that the amino acid sequence of African ostrich BAFF gene and the chicken BAFF gene were found. The homology of the amino acid sequence was the highest, up to 52%, followed by quail (QS BAFF, 51%), duck (D BAFF, 50%), geese (g BAFF, 50%) and Dove (do BAFF, 49%). The analysis of African ostrich BAFF base sequence evolution tree showed that the homology of African ostrich BAFF gene was closest to chicken and quail, followed by duck, goose and dove; the analysis of amino acid sequence evolution tree showed the results. The sequence of amino acids used in the analysis was divided into three branches, one containing all the birds used for analysis, one containing the fish and the other containing all mammals, and the amino acid sequence of the African ostrich BAFF gene was close to that of other birds. These results could be the evolutionary study of the BAFF gene of birds and the molecular biology. The study provided reference.2. The prokaryotic expression of the African ostrich BAFF gene was designed according to the specific primers of the African ostrich BAFF gene sequence obtained from this study. The Eco R I and Hind III enzyme cut loci were added to the downstream primers, and the African ostrich BAFF gene was amplified successfully, and the gene fragment was cloned to P ET-28a prokaryotic expression. PCR, the results of enzyme digestion showed that the recombinant plasmid P ET-Os BAFF. was successfully constructed and transformed into Rosetta (DE3) of Escherichia coli, induced by IPTG, and the expression products were analyzed by SDS-PAGE electrophoresis and Western blotting, and the size of the recombinant protein was 32.2 K D.3, and the African ostrich gene group was determined. Real-time PCR method was used to analyze the expression of M RNA in the African ostrich BAFF gene in 18 tissues. The results showed that BAFF was widely distributed in the tissues detected, but the expression in different tissues was different. The expression of the African ostrich BAFF in the bursa of the Fabricius, the thymus, the spleen and the bone marrow was higher, followed by the liver, The expression of the kidneys, trachea, lungs, stomach and intestines is relatively low in other detected tissues, the lowest is the heart and brain and almost no expression. The difference of the tissue distribution of the African ostrich BAFF gene suggests that its main functions in different tissues may be different from.4, and the biological activity of the recombinant protein of the non ostrich BAFF gene is studied by the biological activity of the ostrich ostrich. The BAFF of dairy animals and other birds is an important factor in the survival of B cells. To verify whether the BAFF protein of the African ostrich can also prolong the survival time of the B cells, the recombinant protein of the African ostrich BAFF gene is purified, and the effect of the recombinant protein in the proliferation of the African ostrich B lymphocyte is determined by MTT. The results showed that when the purified recombinant protein or the purified recombinant protein and the B lymphocyte of the purified recombinant protein and PMA was cultured for 24h, the cell growth was significantly correlated with the concentration of the recombinant protein. When the recombinant protein of 12 mu g/m L was added to the recombinant protein or the reorganizing protein of 16 mu L, and the PMA of 2 mu g/m L, the B lymphocyte grew densely. The test showed that the recombinant protein of the African ostrich BAFF gene could produce a similar result to the proliferation test of the African ostrich's bursa B lymphocyte in vitro, which can also stimulate the survival of the mouse B lymphocytes in vitro. The effect of PBS and BSA on the proliferation of the mouse lymphocyte is similar to that of the African ostrich bursa B lymphocyte proliferation test in vitro. The proliferation.LPS stimulation test showed that the m RNA expression level of the African ostrich BAFF gene was higher than that of the control group after LPS stimulated by LPS. The RNA expression level of the African ostrich BAFF gene m RNA expression level began to rise significantly from the 6 h after LPS stimulation, the maximum value appeared after the stimulation of 24 h, compared with the control group, until 4 after the stimulation. 8 h m RNA expression remained at relatively high level (P0.01).5. The effect of Boron on the expression of African ostrich BAFF gene tissue was to explore the effect of trace element boron on the expression of African ostrich BAFF gene, 48 plumes of 1 days old African ostrich, which were randomly divided into 6 groups, 8 plumes in each group. 0 mg/L (control group) was added to the drinking water, 40, 40, respectively. 40 Mg/L, 80 mg/L, 160 mg/L, 320 mg/L, and 640 mg/L of boric acid were obtained from 1D, 45d and 90d, respectively. The tissue of the bursa, thymus, bone marrow, spleen and other tissues were taken respectively, and the total RNA was extracted as a template. The expression of the group expression of the BAFF gene of African ostrich in different drinking water boron groups was analyzed by fluorescence quantitative PCR. The expression of the BAFF gene m RNA in the 8 tissues increased with the increase of the dosage of boron in the drinking water. When the dosage of boron was added to a certain degree, the expression of BAFF gene reduced the BAFF gene in the bone marrow, spleen, thymus, lung and other tissues when the dosage increased. The expression of the BAFF gene decreased with the.45 day age. The maximum expression of the expression was 160 mg/L test group. The maximum expression of BAFF gene expression in the 3 tissues such as bursa, liver and kidney was 80 mg/L test group, which was significantly higher than that of the control group (P0.05). The maximum expression of BAFF gene in the gastric tissue was 80 mg/L test group, but there was no significant difference between the control group and the control group. The bone marrow, the bursa of Fabricius, the spleen, the thymus, and the kidney were not significantly different from the control group. The maximum expression of BAFF gene expression in the 6 tissues was 80 mg/L test group. The maximum expression of BAFF gene in lung tissue was 160 mg/L test group, which was significantly higher than that of the control group (P0.05), and the maximum value of BAFF gene expression in the gastric tissue was 160 mg/L test group, but there was no significant difference compared with the control group.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S839

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