绵羊白介素1β及其受体拮抗因子全长cDNA克

发布时间：2018-04-20 06:25

本文选题：绵羊 + 白介素1β　；参考：《吉林大学》2015年硕士论文

【摘要】：白介素1β（interleukin1β，IL-1β）和白介素1受体拮抗因子（interleukin1receptor antagonist，IL-1Ra）是白介素1（interleukin1，IL-1）家族的主要成员。IL-1β能够协同其它细胞因子刺激T、B淋巴细胞活化并促进多种炎性物质合成。IL-1Ra与IL-1β三维结构相似，能够与IL-1Ra竞争细胞膜表面的I型白介素1受体（interleukin1receptor I，IL-1RI）。但是IL-1Ra与IL-1RI结合并不激发相关信号，从而能够拮抗IL-1β的生物学功能。IL-1在宿主抵抗病原微生物入侵的过程中起到重要作用，但是IL-1β的过量表达会引起严重的炎症反应及自身免疫疾病。因此，IL-1β和IL-1Ra的平衡对维持机体稳态和预防疾病具有重要意义。研究表明，许多疾病可以引起IL-1β和IL-1Ra特异性的差异表达。通过分析IL-1β和IL-1Ra在疾病中的差异表达模式有望为相关疾病的预防、诊断和治疗工作提供新的思路。布鲁氏菌病(brucellosis)简称“布病”是由布鲁氏菌引起的人畜共患病，其临床特点为长期发热、多汗、关节痛及肝脾肿大等。快捷、可靠的血清学诊断方法，已被广泛用于家畜布鲁氏菌病的检疫，如虎红平板凝集试验。接种布鲁氏菌减毒疫苗是预防布鲁氏菌病的有效措施，但是布鲁氏菌强毒株感染和疫苗免疫都能造成家畜血清学试验阳性。目前，缺乏有效的措施进一步鉴别血清学试验阳性家畜的感染来源，导致无法区别诊断疫苗免疫家畜和布病患病动物，给布病有效净化带来非常大的困难。本课题组前期工作通过构建布鲁氏菌强毒、弱毒株S2感染绵羊白细胞层抑制性消减杂交（SSH）cDNA文库，筛选到差异表达基因IL-1β和IL-1Ra，推测IL-1β和IL-1Ra可能在绵羊感染布鲁氏菌强毒或S2弱毒株过程中存在特异性的差异表达模式。通过揭示这种差异表达模式有望为鉴别血清学虎红平板凝集试验阳性家畜的感染来源提供科学依据。 1.绵羊IL-1β和IL-1Ra全长cDNA克隆、表达及分子特性分析本研究以本课题组构建的布鲁氏菌强毒、S2弱毒株感染绵羊白细胞层SSH文库中获得的IL-1β和IL-1Ra部分基因序列为基础，利用RACE技术，克隆获得绵羊IL-1β和IL-1Ra的全长cDNA序列，其中IL-1β全长1494bp，开放阅读框801bp，编码266个氨基酸残基，预测绵羊IL-1β蛋白分子量为30.6kDa；IL-1Ra全长1228bp，，开放阅读框525bp，编码174个氨基酸残基，预测绵羊IL-1Ra蛋白分子量为19.8kDa。成功构建绵羊IL-1β及IL-1Ra的重组表达质粒pET-30a-IL-1β和pET-28a-IL-1Ra。通过诱导表达和亲合层析纯化技术获得重组表达蛋白orIL-1β及orIL-1Ra。通过生物信息学软件及相关网站预测分析绵羊IL-1β及IL-1Ra核酸与蛋白的分子特性，以及通过胸腺细胞增殖实验证实orIL-1β的生物学活性，进而通过测定orIL-1Ra对IL-1β杀伤鼠黑色素瘤细胞株B16的拮抗作用，证实orIL-1Ra的生物学活性。 2.抗绵羊IL-1β和IL-1Ra单克隆抗体的制备分别应用orIL-1β和orIL-1Ra免疫小鼠。通过细胞融合技术将小鼠B细胞与骨髓瘤细胞SP2/0融合，通过间接ELISA和Western Blot（WB）筛选分泌抗绵羊IL-1β抗体的杂交瘤细胞和分泌抗绵羊IL-1Ra抗体的杂交瘤细胞。抗体亚类鉴定2株杂交瘤细胞株所分泌抗体亚类均为IgG1。通过诱生腹水和G蛋白亲合层析系统分别批量纯化抗绵羊IL-1β和IL-1Ra的2种单克隆抗体。 3.绵羊IL-1β和IL-1Ra时空差异表达特性分析利用本研究制备的单克隆抗体和WB技术，分析正常绵羊不同组织器官中IL-1β及IL-1Ra蛋白的组织特异性表达特性。发现IL-1β在正常绵羊的脾脏中含量最高，在白细胞中含量最低；IL-1Ra在正常绵羊肌肉中含量最高，而在肺脏中含量最低。通过大肠杆菌、单核细胞增生性李斯特氏菌（李氏杆菌）、沙门氏菌、布鲁氏菌S2弱毒株和灭活布鲁氏菌弱毒株S2菌体侵染绵羊原代外周血白细胞，对侵染后不同时间点细胞培养上清液中IL-1β和IL-1Ra的含量进行测定。大肠杆菌、李氏杆菌、沙门氏菌、布鲁氏菌弱毒株S2侵染绵羊外周血白细胞后细胞培养上清中IL-1β及IL-1Ra的含量均逐渐上升。灭活布鲁氏菌弱毒株S2组白细胞培养上清液中2种细胞因子的相对含量均呈现先升高后下降的趋势。利用荧光定量PCR技术分析布鲁氏菌强毒和弱毒株S2感染绵羊后IL-1β及IL-1Ra基因在外周血白细胞层中转录水平的差异表达模式。IL-1β基因在强毒组和弱毒组白细胞层中的表达均呈先下调后上调的表达趋势，强毒组在感染第14天时表达量最低，感染40天时表达量最高。在弱毒组感染7天时表达量最低，感染21天时表达量最高。IL-1Ra基因在强毒组和弱毒组中的表达都呈现出上调表达的趋势。应用WB技术分析布鲁氏菌强毒和弱毒株S2感染绵羊后IL-1β及IL-1Ra在血浆中的含量变化。IL-1β在强毒组及弱毒组血浆中的含量均随着感染时间的延长先升高后下降。强毒组IL-1β升高水平高于弱毒组。IL-1Ra在强毒组及弱毒组血浆中的含量均在感染后40天明显升高，而且在强毒组升高的水平高于弱毒组。本研究以绵羊IL-1β及IL-1Ra为研究对象，克隆其全长cDNA，利用原核表达系统表达并纯化基因工程重组蛋白，制备其单克隆抗体，分析IL-1β和IL-1Ra的分子特性及其在正常绵羊和细菌侵染状态下时空差异表达模式，为深入研究布病感染与免疫区别诊断生物标示分子提供科学依据。
[Abstract]:Interleukin - 1尾 ( IL - 1尾 ) and interleukin - 1 receptor antagonist ( IL - 1Ra ) are the main members of interleukin - 1 ( IL - 1 ) family . IL - 1Ra can cooperate with other cytokines to stimulate T and B lymphocytes to activate and promote the synthesis of various inflammatory substances . IL - 1Ra is similar to the three - dimensional structure of IL - 1尾and can compete with IL - 1Ra to compete with IL - 1Ra to compete with type I interleukin 1 receptor ( IL - 1RI ) on the surface of cell membrane . IL - 1尾 and IL - 1Ra play an important role in maintaining homeostasis and preventing diseases . Therefore , the expression pattern of IL - 1尾and IL - 1Ra is expected to provide a new idea for the prevention , diagnosis and treatment of related diseases .

The results of this study have been widely used in quarantine such as chronic fever , hyperhidrosis , arthralgia , hepatosplenostasis and so on . It has been widely used in quarantine of domestic animals , such as tiger red plate agglutination test .

1 . Cloning , Expression and Molecular Characterization of Full - length cDNA of IL - 1尾 and IL - 1Ra in Sheep

The full - length cDNA sequence of IL - 1尾 and IL - 1Ra in sheep white blood cell layer SSH library was obtained by RACE technique . The cDNA sequence of IL - 1尾 and IL - 1Ra was cloned by RACE . The length of IL - 1尾was 1494bp , the open reading frame was 801bp , and 266 amino acid residues were encoded .
The recombinant expression plasmid pET - 30a - IL - 1尾and pET - 28a - IL - 1Ra were successfully constructed .

2 . Preparation of Anti - sheep IL - 1尾 and IL - 1Ra Monoclonal Antibodies

The hybridoma cells secreting anti - sheep IL - 1尾antibody and hybridoma cells secreting anti - sheep IL - 1Ra antibodies were screened by indirect ELISA and Western Blot ( WB ) . Two monoclonal antibodies against sheep IL - 1尾 and IL - 1Ra were purified by indirect ELISA and Western Blot ( WB ) .

3 . Analysis of Temporal and Spatial Differential Expression of IL - 1尾 and IL - 1Ra in Sheep

The specific expression of IL - 1尾 and IL - 1Ra in different tissues and organs of normal sheep was analyzed by using the monoclonal antibody and WB technique prepared by the study . It was found that the content of IL - 1尾 in the spleen of normal sheep was the highest , and the lowest in white blood cells .
IL - 1Ra is the highest in normal sheep muscle , while the lowest in lung .

The levels of IL - 1尾 and IL - 1Ra in cultured supernatant of sheep were determined by means of E . coli , M . li , S . S . S . , S.typhimurium , S2 attenuated strain and inactivated brucella strain S2 . The levels of IL - 1尾 and IL - 1Ra in the supernatant of culture supernatant were gradually increased after infection .

The expression of IL - 1尾and IL - 1Ra in plasma of virulent and weakly virulent groups was lowest . The levels of IL - 1Ra and IL - 1Ra in plasma of virulent and attenuated groups were higher than those in the weak group . The levels of IL - 1Ra and IL - 1Ra in plasma of virulent and attenuated groups were higher than those in the group of weak toxin .

In order to study the molecular characteristics of IL - 1尾 and IL - 1Ra and the expression pattern of space - time difference in normal sheep and bacteria infection , the molecular characteristics of IL - 1尾 and IL - 1Ra and the expression pattern of space - time difference in normal sheep and bacteria were analyzed .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

【参考文献】