非洲鸵鸟BAFF的克隆表达及饮水硼对其表达的影响

发布时间：2018-06-09 06:50

本文选题：非洲鸵鸟 + B淋巴细胞刺激因子　；参考：《华中农业大学》2016年博士论文

【摘要】：B细胞刺激因子(B cell activating factor,BAFF)是肿瘤坏死因子(tumor necrosis factor,TNF)家族中的主要成员之一,是具有维持B细胞功能稳定作用的重要细胞因子。BAFF与T细胞的活化、B淋巴细胞的发育及体液免疫等直接相关。BAFF可以使尚未完全成熟的B细胞分化成更加成熟的B细胞,同时BAFF还可以延长B细胞的存活、促进抗体的产生,BAFF因其在B细胞发育及自身免疫病中起非常重要的作用而日益受人关注和重视。非洲鸵鸟因其生长速度快,适应性广,饲料转化率高,近年来其养殖规模稳步发展。但是在非洲鸵鸟的养殖过程中,3月龄以内的雏鸵鸟对多种病原的易感性较强,非常容易发生各类疾病,给非洲鸵鸟养殖业造成巨大的经济损失。硼(Boron,B)是一种微量元素,广泛分布于自然界。硼具有多种生物学作用。近年的研究表明,硼可能是某些动物生长发育和新陈代谢过程中不可或缺的微量元素。对动物的大量研究发现硼可以影响机体的免疫功能,生理剂量的硼具有止痛、抗炎和增强免疫等作用,较高剂量的硼具有毒性作用,可导致免疫器官发育受阻和免疫功能低下。本研究以非洲鸵鸟为对象,参考已有的鸟类BAFF基因序列设计引物,扩增鸵鸟BAFF基因,通过基因克隆、原核表达和荧光定量PCR等方法研究BAFF基因在各免疫器官内的表达及其蛋白的生物学活性。同时通过在饮水中人工添加不同剂量的硼,利用实时荧光定量PCR等方法检测分析硼对BAFF基因在非洲雏鸵鸟不同组织中表达情况的影响,为探究硼的营养作用和免疫毒性作用奠定基础,为非洲鸵鸟的不同病原免疫防御机制的研究提供有价值的参考,同时也可为非洲鸵鸟及其它鸟类的疾病防治提供科学的依据。主要研究内容与结果如下:1、非洲鸵鸟BAFF基因的克隆与序列分析参考GenBank中公布的其它禽类BAFF的基因序列设计引物,以非洲鸵鸟法氏囊总RNA为模板,经RT-PCR扩增,成功扩增出非洲鸵鸟BAFF基因全序列。所得序列用MEGA 5.05软件和Signal P 4.1 server软件对其碱基序列和氨基酸序列进行同源性分析和进化树分析。结果表明:非洲鸵鸟BAFF基因全长867 bp,编码288个氨基酸;同源性分析发现,非洲鸵鸟BAFF基因氨基酸序列与鸡BAFF基因氨基酸序列的同源性最高,达52%,其次是鹌鹑(qs BAFF,51%)、鸭(d BAFF,50%)、鹅(g BAFF,50%)和鸽子(do BAFF,49%);非洲鸵鸟BAFF基因碱基序列进化树分析显示非洲鸵鸟BAFF基因与鸡、鹌鹑的同源性最近,其次是鸭、鹅和鸽子;氨基酸序列进化树分析结果显示分析所用的氨基酸序列分为三支,一支包含了分析所用的所有鸟类,一支包含了所用的鱼类,另一支包含了所有的哺乳动物,非洲鸵鸟BAFF基因氨基酸序列与其它鸟类的同源性较近。这些结果可为鸟类BAFF基因的进化研究以及分子生物学研究提供参考。2、非洲鸵鸟BAFF基因的原核表达根据本研究克隆获得的非洲鸵鸟BAFF基因序列设计特异性引物,分别在上、下游引物中加入Eco R I和Hind III酶切位点,成功扩增出非洲鸵鸟BAFF基因后,将该基因片段克隆至p ET-28a原核表达载体;PCR、酶切鉴定结果表明成功构建了阳性重组表达质粒p ET-Os BAFF。将该重组质粒转化入大肠杆菌Rosetta(DE3)中,经IPTG诱导表达,将表达产物进行SDS-PAGE电泳分析、Western blotting验证,最终确定该重组蛋白大小为32.2 k D。3、非洲鸵鸟BAFF基因的组织表达研究采用real-time PCR方法分析了非洲鸵鸟BAFF基因在18种组织中的m RNA表达情况。结果显示:BAFF广泛分布于所检测的组织中,但在不同组织中的表达量有差异。非洲鸵鸟BAFF在法氏囊、胸腺、脾脏和骨髓等组织中表达量较高,其次是肝、肾脏、气管、肺、胃和肠,在其它检测的组织中的表达量相对较低,最低的是心脏和脑,几乎无表达。非洲鸵鸟BAFF基因组织分布的差异提示其在不同组织中所发挥的主要功能可能有所不同。4、非洲鸵鸟BAFF基因重组蛋白的生物学活性研究哺乳动物和其它鸟类的BAFF是B细胞存活的重要影响因子。为验证非洲鸵鸟的BAFF蛋白是否也可以延长B细胞的存活时间。对表达获得的非洲鸵鸟BAFF基因重组蛋白进行纯化,利用MTT实验法测定该重组蛋白在非洲鸵鸟B淋巴细胞增殖中的作用。结果显示:添加了纯化的重组蛋白或添加纯化的重组蛋白和PMA的试验组的B淋巴细胞培养24h时,其细胞生长情况与重组蛋白的添加浓度有明显的相关性,当添加12μg/m L的重组蛋白或者16μg/m L的重组蛋白和2μg/m L的PMA时,B淋巴细胞生长密度达到最大值,而对照组添加PBS及BSA组,其B淋巴细胞均无明显增多。对小鼠淋巴细胞增殖的影响试验表明,非洲鸵鸟BAFF基因重组蛋白在体外可以产生与非洲鸵鸟法氏囊B淋巴细胞增殖试验类似的结果,同样可以刺激小鼠B淋巴细胞存活/增殖。LPS刺激实验结果表明,非洲鸵鸟BAFF基因m RNA表达水平在法氏囊淋巴细胞受LPS刺激后的6、12、24及48小时后均高于对照组。非洲鸵鸟BAFF基因m RNA表达水平自LPS刺激后6 h开始大幅上调,最大值出现在刺激后24 h;与对照组相比,直到刺激后48 h其m RNA表达一直保持在相对较高水平(p0.01)。5、饮水硼对非洲鸵鸟BAFF基因组织表达的影响为探究微量元素硼对非洲鸵鸟BAFF基因组织表达的影响,取健康1日龄非洲鸵鸟48羽,将其随机分为6组,每组8羽。分别在饮水中添加0 mg/L(对照组),40 mg/L,80 mg/L,160 mg/L,320 mg/L,640 mg/L剂量的硼酸,分别于1d,45d和90d取材,分别取法氏囊、胸腺、骨髓、脾等组织,提取总RNA作为模板,采用荧光定量PCR法分析不同饮水硼组非洲鸵鸟BAFF基因的组织表达情况。结果发现:非洲鸵鸟BAFF基因m RNA的组织表达与饮水中硼的添加剂量相关。低剂量时,所测8种组织中BAFF基因m RNA的表达随饮水中硼添加剂量的增加而升高;当硼添加剂量到一定程度时,随着添加剂量的增大,BAFF基因的表达反而降低。45日龄时,骨髓、脾、胸腺、肺等4种组织中BAFF基因表达的最大值为160 mg/L试验组,法氏囊、肝、肾等3种组织中BAFF基因表达的最大值为80 mg/L试验组,均显著高于对照组(P0.05);胃组织中BAFF基因表达的最大值为80 mg/L试验组,但与对照组相比差异性不显著。90日龄时,骨髓、法氏囊、肝、脾、胸腺、肾等6种组织中BAFF基因表达的最大值为80 mg/L试验组,肺组织中BAFF基因表达的最大值为160 mg/L试验组,均显著高于对照组(P0.05);胃组织中BAFF基因表达的最大值为160 mg/L试验组,但与对照组相比无显著性差异。
[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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S839

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