本文選題：N-甲基-D-天門(mén)冬氨酸受體 + 抗原表位��； 參考：《瀘州醫(yī)學(xué)院》2010年碩士論文

【摘要】： 目的：N-甲基-D-天門(mén)冬氨酸受體(N-methyl-D-aspartate Receptor, NMDAR, NR)與興奮性突觸傳遞、突觸可塑性、學(xué)習(xí)和記憶等許多中樞活動(dòng)密切相關(guān),其過(guò)度激活所介導(dǎo)的興奮毒性與機(jī)體應(yīng)激、藥物成癮性、疼痛、腦組織繼發(fā)損傷等有密切關(guān)系。通過(guò)選擇性干預(yù)NR活動(dòng),可能為相關(guān)疾病提供有效的治療手段。已有的NR拮抗劑或阻斷劑均為人工合成的小分子藥物,容易彌散通過(guò)血腦屏障,但作用選擇性低,常引起意識(shí)模糊、焦慮不安、幻覺(jué)等嚴(yán)重毒副作用,且存在難以超早期用藥的問(wèn)題,難以進(jìn)入臨床。NR1是NR的功能亞單位,預(yù)測(cè)NR1抗原表位,制備N(xiāo)R1口服疫苗是超早期干預(yù)機(jī)體應(yīng)激、腦損傷、藥物成癮、疼痛等的可行方法之一。轉(zhuǎn)鐵蛋白受體(Transferrin receptor,TfR)抗體可能可以攜帶NR1抗原通過(guò)血腦屏障,從而使得NR1抗原能夠更好的在中樞神經(jīng)系統(tǒng)發(fā)揮作用。因此,本研究旨在用噬菌體展示技術(shù)預(yù)測(cè)小鼠NR1抗原表位,選擇小鼠TfR構(gòu)建NR1-TfR融合蛋白真核表達(dá)載體,為NR1口服疫苗的制備做好前期準(zhǔn)備。 方法：①用小鼠NR1單克隆抗體(Monclone antibody, mAb)免疫親和篩選以融合蛋白形式表達(dá)在絲狀噬菌體M13外殼蛋白Ⅲ上的隨機(jī)十二肽,經(jīng)過(guò)三輪的生物篩淘后,從第三輪洗脫物中挑取所有56個(gè)噬菌體克隆,提取噬菌體單鏈DNA并測(cè)序。②從Genebank查到小鼠TfR編碼序列(Coding sequence,CDS),編號(hào)為GenBank NP_035768,將此序列和篩選所得NR1抗原表位優(yōu)勢(shì)序列,通過(guò)設(shè)計(jì)引物、聚合酶鏈反應(yīng)(Polymerase chain reaction, PCR)合成融合表達(dá)基因序列(將該基因片段命名為NR1-TfR)并連接到質(zhì)粒載體pUC57(將該質(zhì)粒命名為pUC57-NRl-TfR)。③通過(guò)限制性?xún)?nèi)切酶KpnI和XbaI雙酶切質(zhì)粒pUC57-NR1-TfR獲得NR1-TfR片段,并連接到真核表達(dá)載體pcDNA3.1中,然后用氯化鈣法轉(zhuǎn)化到大腸桿菌DH5α,氨芐青霉素抗性篩選獲得陽(yáng)性克隆,擴(kuò)增后提取質(zhì)粒,進(jìn)行KpnI和XbaI雙酶切后電泳,并進(jìn)行生物測(cè)序鑒定。結(jié)果：經(jīng)過(guò)3輪篩選后能與NR1單克隆抗體特異性結(jié)合的噬菌體得到了有效富集,DNA測(cè)序結(jié)果表明56個(gè)單克隆噬菌體中有54個(gè)表達(dá)為同一個(gè)氨基酸序列：DDWVISTQSLKS,其余兩個(gè)單克隆噬菌體表達(dá)的氨基酸序列分別為：HSSHTSNTLPSV和TNTPPSQRVHLS。酶切、電泳證實(shí)NR1-TfR片段被成功的克隆到質(zhì)粒載體pcDNA3.1中,測(cè)序結(jié)果證明重組質(zhì)粒載體插入序列與設(shè)計(jì)的靶基因片段完全一致。結(jié)論：從噬菌體展示的隨機(jī)十二肽庫(kù)中成功篩選到了能與NR1特異性抗體結(jié)合的短肽DDWVISTQSLKS,該肽是NR1單克隆抗體結(jié)合的優(yōu)勢(shì)序列,可能模擬了天然抗原的某個(gè)表位。成功構(gòu)建該優(yōu)勢(shì)序列與小鼠TfR真核表達(dá)質(zhì)粒pcDNA3.1-NR1-TfR,為NR1口服疫苗的制備及其后續(xù)實(shí)驗(yàn)奠定了基礎(chǔ)。
[Abstract]:Objective: N-methyl-D-aspartate Receptor, NMDAR, NR) is closely related to excitatory synaptic transmission, synaptic plasticity, learning and memory, and its excitotoxicity mediated by excessive activation is associated with stress, drug addiction and pain. There is a close relationship between brain secondary injury and so on. Selective intervention in NR activity may provide effective treatment for related diseases. The existing NR antagonists or blockers are synthetic small molecular drugs, which are easily dispersed through the blood-brain barrier, but have low selectivity, and often cause serious side effects such as confusion, anxiety, hallucination and other serious side effects. Moreover, it is difficult to use drugs in the very early stage and difficult to enter the clinic. NR1 is the functional subunit of NR. It is one of the feasible methods to predict the epitopes of NR1 antigen and prepare oral NR1 vaccine to interfere with stress, brain injury, drug addiction, pain and so on. Transferrin receptor TfR antibodies may carry NR1 antigens across the blood-brain barrier, thus enabling NR1 antigens to play a better role in the central nervous system. Therefore, the purpose of this study was to predict mouse NR1 epitopes by phage display technique, select mouse TfR to construct eukaryotic expression vector of NR1-TfR fusion protein, and prepare for the preparation of oral NR1 vaccine. Methods using mouse NR1 monoclonal antibody Monclone antibody (mAb), we screened the random dodecapeptide expressed on the filamentous phage M13 coat protein 鈪，

本文編號(hào)：1822651