NR1抗原与TfR融合蛋白真核表达质粒的构建

发布时间：2018-04-30 02:22

  本文选题：N-甲基-D-天门冬氨酸受体 + 抗原表位　； 参考：《泸州医学院》2010年硕士论文

【摘要】： 目的：N-甲基-D-天门冬氨酸受体(N-methyl-D-aspartate Receptor, NMDAR, NR)与兴奋性突触传递、突触可塑性、学习和记忆等许多中枢活动密切相关,其过度激活所介导的兴奋毒性与机体应激、药物成瘾性、疼痛、脑组织继发损伤等有密切关系。通过选择性干预NR活动,可能为相关疾病提供有效的治疗手段。已有的NR拮抗剂或阻断剂均为人工合成的小分子药物,容易弥散通过血脑屏障,但作用选择性低,常引起意识模糊、焦虑不安、幻觉等严重毒副作用,且存在难以超早期用药的问题,难以进入临床。NR1是NR的功能亚单位,预测NR1抗原表位,制备NR1口服疫苗是超早期干预机体应激、脑损伤、药物成瘾、疼痛等的可行方法之一。转铁蛋白受体(Transferrin receptor,TfR)抗体可能可以携带NR1抗原通过血脑屏障,从而使得NR1抗原能够更好的在中枢神经系统发挥作用。因此,本研究旨在用噬菌体展示技术预测小鼠NR1抗原表位,选择小鼠TfR构建NR1-TfR融合蛋白真核表达载体,为NR1口服疫苗的制备做好前期准备。 方法：①用小鼠NR1单克隆抗体(Monclone antibody, mAb)免疫亲和筛选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机十二肽,经过三轮的生物筛淘后,从第三轮洗脱物中挑取所有56个噬菌体克隆,提取噬菌体单链DNA并测序。②从Genebank查到小鼠TfR编码序列(Coding sequence,CDS),编号为GenBank NP_035768,将此序列和筛选所得NR1抗原表位优势序列,通过设计引物、聚合酶链反应(Polymerase chain reaction, PCR)合成融合表达基因序列(将该基因片段命名为NR1-TfR)并连接到质粒载体pUC57(将该质粒命名为pUC57-NRl-TfR)。③通过限制性内切酶KpnI和XbaI双酶切质粒pUC57-NR1-TfR获得NR1-TfR片段,并连接到真核表达载体pcDNA3.1中,然后用氯化钙法转化到大肠杆菌DH5α,氨苄青霉素抗性筛选获得阳性克隆,扩增后提取质粒,进行KpnI和XbaI双酶切后电泳,并进行生物测序鉴定。结果：经过3轮筛选后能与NR1单克隆抗体特异性结合的噬菌体得到了有效富集,DNA测序结果表明56个单克隆噬菌体中有54个表达为同一个氨基酸序列：DDWVISTQSLKS,其余两个单克隆噬菌体表达的氨基酸序列分别为：HSSHTSNTLPSV和TNTPPSQRVHLS。酶切、电泳证实NR1-TfR片段被成功的克隆到质粒载体pcDNA3.1中,测序结果证明重组质粒载体插入序列与设计的靶基因片段完全一致。结论：从噬菌体展示的随机十二肽库中成功筛选到了能与NR1特异性抗体结合的短肽DDWVISTQSLKS,该肽是NR1单克隆抗体结合的优势序列,可能模拟了天然抗原的某个表位。成功构建该优势序列与小鼠TfR真核表达质粒pcDNA3.1-NR1-TfR,为NR1口服疫苗的制备及其后续实验奠定了基础。
[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，

本文编号：1822651