本文關(guān)鍵詞：人Toll樣受體9基因克隆及真核重組載體的構(gòu)建　出處：《吉林大學》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： TLRs 轉(zhuǎn)化 RT-PCR 基因克隆

【摘要】：Toll樣受體9(Toll-Like Receptor9,TLR9),是重要的模式識別受體,識別一組具有特定結(jié)構(gòu)的寡核苷酸序列CpG,其廣泛地存在于病原體中。TLR9在識別病原體和激活天然免疫方面起著非常重要的作用。激活的TLR9不但能夠誘導天然免疫應答,并且有助于特異性免疫反應的發(fā)生。TLR9能夠介導CpG對多種免疫細胞的激活,例如巨噬細胞、B細胞、DC細胞；由此,將使細胞表面CD40、CD80、CD86等共刺激分子以及MHCII類分子表達增加,而且促使細胞因子IL-6,、IL-12,、TNF、IFN-γ、IFN-α等的分泌,從而誘導Th1型免疫應答。除參與誘導的細胞活化,TLR9還能通過延長活化DC細胞的壽命,抑制細胞凋亡,穩(wěn)定Th1型免疫應答。通過CpG與TLR9的作用,DC細胞等抗原提呈細胞能夠交叉提呈外源性抗原,從而交叉激活CD8+細胞。TLR9過度激活,反而會抑制DCs成熟,使調(diào)節(jié)性T細胞增多。TLR9是連接獲得性免疫和天然免疫的橋梁。TLR9基因具有多態(tài)性,會影響TLR9受體蛋白的表達和功能活性,使機體的天然免疫功能受限,與機體對感染性疾病的易感性密切相關(guān)。 本研究從人外周血細胞中提取總RNA,利用逆轉(zhuǎn)錄PCR (Reverse transcript-polymerase chain reaction, RT-PCR)擴增TLR9基因的cDNA序列.通過TA克隆,將TLR9插入PMD18T。通過藍白篩選,篩選出陽性克隆。應用亞克隆技術(shù),將TLR9基因與pcDNA3.0載體重組,經(jīng)質(zhì)粒提取初篩、雙酶切鑒定正確構(gòu)建了真核表達質(zhì)粒pcDNA3.0-TLR9。將重組基因轉(zhuǎn)化入感受態(tài)細胞JM109中,得到重組質(zhì)粒的工程菌。測序結(jié)果表明,與Genbank的TLR9比對結(jié)果相同,有四處堿基發(fā)生置換。其中1635bp和3036bp處,均為簡并堿基,編碼的氨基酸不變。209bp處,t置換為c,編碼的氨基酸由色氨酸變?yōu)榻z氨酸。3057bp處,c置換為g,編碼的組氨酸變?yōu)楣劝滨０�。本研究得出的結(jié)果能夠進一步研究TLR9及其配體的表達和生物學活性,建立穩(wěn)定表達TLR9的細胞系。為與TLR9相關(guān)的免疫及炎癥方面的疾病研究和治療提供新的思路。
[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.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前2條

1 黃巧茹;;TLR9,先天免疫中重要的抗微生物受體[J];中山大學研究生學刊(自然科學、醫(yī)學版);2007年03期

2 KM Lammers;S Ouburg;SA Morré;JBA Crusius;P Gionchetti;F Rizzello;C Morselli;E Caramelli;R Conte;G Poggioli;M Campieri;AS Pe濼a;;Combined carriership of TLR9 -1237C and CD14 -260T alleles enhances the risk of developing chronic relapsing pouchitis[J];World Journal of Gastroenterology;2005年46期

，

本文編號：1376716