本文關(guān)鍵詞：pEGFP-Cl-MyD88真核表達(dá)載體的構(gòu)建及其在HEK293細(xì)胞中的表達(dá)　出處：《華中師范大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 水牛 髓樣分化因子88 增強(qiáng)型綠色熒光蛋白 融合蛋白 穩(wěn)定細(xì)胞系

【摘要】：Toll樣受體(Toll-like receptors, TLRs)是新近發(fā)現(xiàn)的模式識(shí)別受體,該家族通過識(shí)別病原體相關(guān)分子模式pathogen associated molecular patterns, PAMP)激活免疫細(xì)胞,在天然免疫防御病原體中起著重要作用。髓樣分化因子88(myeloid differentiation factor88, MyD88)作為一種細(xì)胞內(nèi)信號(hào)接頭蛋白,在TLR家族和白介素1受體(interleukin1receptor, IL-1R)家族信號(hào)通路中發(fā)揮關(guān)鍵作用。MyD88蛋白具有3個(gè)功能結(jié)構(gòu)域：N端的死亡結(jié)構(gòu)域(death domain, DD)、中間區(qū)域(intermediate domain, ID)及C端的Toll樣受體和白介素1受體相關(guān)結(jié)構(gòu)域(TLR/IL-1R related domain, TIR),其中MyD88蛋白的TIR結(jié)構(gòu)域能與TLRs和IL-1Rs的TIR結(jié)構(gòu)域相結(jié)合,從而介導(dǎo)信號(hào)向下游傳導(dǎo)。MyD88是重要的信號(hào)轉(zhuǎn)接蛋白,其介導(dǎo)的信號(hào)通路與腸炎、動(dòng)脈粥樣硬化、心肌肥大、癌癥等在內(nèi)的多種疾病的發(fā)生和發(fā)展過程有著密切的聯(lián)系。MyD88的結(jié)構(gòu)和功能研究將為這些疾病的治療提供新的方向和思路。本研究以水牛外周血白細(xì)胞為材料,提取總RNA,通過RT-PCR的方法擴(kuò)增MyD88基因全長(zhǎng)編碼序列,將其連接至pEGFP-Cl質(zhì)粒,構(gòu)建(?)EGFP-Cl-MyD88真核重組表達(dá)載體,重組載體經(jīng)菌落PCR、酶切和測(cè)序鑒定后,在脂質(zhì)體的介導(dǎo)下轉(zhuǎn)染進(jìn)HEK293細(xì)胞中,使其發(fā)生瞬時(shí)表達(dá),并通過G418抗性篩選,構(gòu)建穩(wěn)定表達(dá)EGFP-MyD88融合蛋白的HEK293細(xì)胞系。結(jié)果表明：轉(zhuǎn)染48h后,綠色熒光蛋白在轉(zhuǎn)染重組質(zhì)粒的細(xì)胞中呈顆粒狀散在分布,而在轉(zhuǎn)染空質(zhì)粒的細(xì)胞中均勻分布；Western Blot結(jié)果顯示在62kDa處出現(xiàn)一條特異性抗體結(jié)合條帶；經(jīng)過G418的持續(xù)篩選,獲得了穩(wěn)定表達(dá)目的蛋白的陽性克隆,FCM分析結(jié)果顯示,轉(zhuǎn)染pEGFP-Cl空質(zhì)粒的細(xì)胞系陽性率達(dá)93.35%,轉(zhuǎn)染重組質(zhì)粒的細(xì)胞系陽性率達(dá)84.33%,表明成功構(gòu)建了穩(wěn)定表達(dá)MyD88的HEK293細(xì)胞系。以上結(jié)果為針對(duì)MyD88分子的疾病治療及其功能研究奠定了基礎(chǔ)。
[Abstract]:Toll-like receptor (TLRs) is a newly discovered pattern recognition receptor. The family activates immune cells by recognizing the pathogen-associated molecular pattern pathogen associated molecular patterns (PAMPs). Myeloid differentiation factor88, myeloid differentiation factor 88, plays an important role in innate immune defense. MyD88) is a intracellular signaling junction protein in the TLR family and interleukin-1 receptor. IL-1R family signaling pathway plays a key role. MyD88 protein has three functional domains: N-terminal death domain death domain (DDD). Intermediate domain. Toll like receptor and interleukin-1 receptor related domain TLR / IL-1R related domain (TIRs). The TIR domain of MyD88 protein can bind to the TIR domain of TLRs and IL-1Rs, which mediates signal transduction downstream. MyD88 is an important signal transfer protein. It mediates signaling pathways associated with enteritis, atherosclerosis, and myocardial hypertrophy. The structure and function of MyD88 are closely related to the occurrence and development of many diseases, such as cancer, which will provide a new direction and thought for the treatment of these diseases. For material. Total RNAs were extracted, the full-length coding sequence of MyD88 gene was amplified by RT-PCR, and ligated into pEGFP-Cl plasmid. EGFP-Cl-MyD88 eukaryotic expression vector was transfected into HEK293 cells with liposome mediated by restriction endonuclease digestion and sequencing. The HEK293 cell lines expressing EGFP-MyD88 fusion protein stably were constructed by G418 resistance screening. The results showed that the cells were transfected for 48 h. Green fluorescent protein (GFP) was distributed in the cells transfected with recombinant plasmids and distributed uniformly in the cells transfected with empty plasmids. Western Blot showed a specific antibody binding band at 62 kDa. The results of FCM analysis showed that the positive rate of cell lines transfected with empty plasmid of pEGFP-Cl was 93.35%. The positive rate of cell line transfected with recombinant plasmid was 84.33%. The results showed that the HEK293 cell lines stably expressing MyD88 were successfully constructed, which laid a foundation for the study of disease treatment and function of MyD88 molecules.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329;Q78

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張婷;鹽藻促有絲分裂活化蛋白激酶基因DsMAPK的克隆與表達(dá)[D];大連海洋大學(xué);2013年

，

本文編號(hào)：1375722