人Toll样受体9基因克隆及真核重组载体的构建

发布时间：2018-01-04 02:44

本文关键词：人Toll样受体9基因克隆及真核重组载体的构建　出处：《吉林大学》2011年硕士论文　论文类型：学位论文

【摘要】：Toll样受体9(Toll-Like Receptor9,TLR9),是重要的模式识别受体,识别一组具有特定结构的寡核苷酸序列CpG,其广泛地存在于病原体中。TLR9在识别病原体和激活天然免疫方面起着非常重要的作用。激活的TLR9不但能够诱导天然免疫应答,并且有助于特异性免疫反应的发生。TLR9能够介导CpG对多种免疫细胞的激活,例如巨噬细胞、B细胞、DC细胞；由此,将使细胞表面CD40、CD80、CD86等共刺激分子以及MHCII类分子表达增加,而且促使细胞因子IL-6,、IL-12,、TNF、IFN-γ、IFN-α等的分泌,从而诱导Th1型免疫应答。除参与诱导的细胞活化,TLR9还能通过延长活化DC细胞的寿命,抑制细胞凋亡,稳定Th1型免疫应答。通过CpG与TLR9的作用,DC细胞等抗原提呈细胞能够交叉提呈外源性抗原,从而交叉激活CD8+细胞。TLR9过度激活,反而会抑制DCs成熟,使调节性T细胞增多。TLR9是连接获得性免疫和天然免疫的桥梁。TLR9基因具有多态性,会影响TLR9受体蛋白的表达和功能活性,使机体的天然免疫功能受限,与机体对感染性疾病的易感性密切相关。本研究从人外周血细胞中提取总RNA,利用逆转录PCR (Reverse transcript-polymerase chain reaction, RT-PCR)扩增TLR9基因的cDNA序列.通过TA克隆,将TLR9插入PMD18T。通过蓝白筛选,筛选出阳性克隆。应用亚克隆技术,将TLR9基因与pcDNA3.0载体重组,经质粒提取初筛、双酶切鉴定正确构建了真核表达质粒pcDNA3.0-TLR9。将重组基因转化入感受态细胞JM109中,得到重组质粒的工程菌。测序结果表明,与Genbank的TLR9比对结果相同,有四处碱基发生置换。其中1635bp和3036bp处,均为简并碱基,编码的氨基酸不变。209bp处,t置换为c,编码的氨基酸由色氨酸变为丝氨酸。3057bp处,c置换为g,编码的组氨酸变为谷氨酰氨。本研究得出的结果能够进一步研究TLR9及其配体的表达和生物学活性,建立稳定表达TLR9的细胞系。为与TLR9相关的免疫及炎症方面的疾病研究和治疗提供新的思路。
[Abstract]:Toll like receptor 9 (Toll-Like, Receptor9, TLR9) are important pattern recognition receptors, CpG oligonucleotide sequences with specific structure identification of a group, which widely exists in the pathogen.TLR9 in pathogen recognition and activation of innate immunity plays a very important role. The activation of TLR9 can not only induce innate immune responses, and contribute to the specific immune reaction to.TLR9 mediated CpG activation of many immune cells such as macrophages, B cells and DC cells; thus, the cell surface CD40, CD80, CD86 and costimulatory molecules and MHCII molecules and promote increased expression of cytokines IL-6, IL-12, and TNF. IFN-, gamma, IFN- secretion, induce Th1 type immune response. In addition to participating in the TLR9 induced cell activation, but also through the activation of DC cells prolong life, inhibit apoptosis, stable Th1 immune response by CpG and TLR9. The role of DC cells and antigen-presenting cells capable of cross presentation of exogenous antigens, which cross over activation of.TLR9 activated CD8+ cells, but could inhibit DCs maturation, the regulatory T cells increased.TLR9 is a bridge connecting the.TLR9 gene acquired immunity and innate immunity with polymorphism can affect the expression and function of TLR9 receptor activity protein, natural immune function is limited in the body, and is closely related to susceptibility to infectious diseases.
The extraction of total RNA from human peripheral blood cells, using reverse transcription PCR (Reverse transcript-polymerase chain reaction, RT-PCR) cDNA sequence of TLR9 gene was amplified by TA. Cloning, insert the TLR9 into the PMD18T. through the blue white screening, the positive clones were screened. Using subcloning technique, TLR9 gene and pcDNA3.0 carrying weight group. After Plasmid Extraction screening, enzyme cuuing constructed eukaryotic expression plasmid of recombinant pcDNA3.0-TLR9. gene was transformed into competent cell JM109, engineering bacteria obtained recombinant plasmid. The sequencing result showed that the ratio of TLR9 and Genbank are the same, there are four base replacement. The 1635bp and 3036bp, are simple and BP, encoding amino acid replacement for t invariant.209bp, C, encoding amino acid from tryptophan to serine.3057bp, C replacement for G, encoding histidine into glutamine. The results of this study We can further study the expression and biological activity of TLR9 and its ligands, and establish a cell line stably expressing TLR9. This will provide new ideas for TLR9 related research and treatment of immune and inflammatory diseases.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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