NR1-TFR重组表位疫苗的制备及其在真核细胞中的表达

发布时间：2018-09-12 13:22

【摘要】：目的：N—甲基—D—天冬氨酸受体(N-Methyl-D-Asparate Receptor, NR)广泛参与应激、脑损伤、药物成瘾、疼痛等病理生理过程。选择性阻断NR活性可能成为相关疾病的治疗手段。现有的NR拮抗剂或阻断剂均为人工合成的小分子药物,由于作用选择性低常引起幻觉、焦虑不安、意识模糊等严重毒副作用,并存在难以超早期用药的问题。NR1是NR的功能亚单位,具有NR1的一切电生理学特性和药理学活性。NR1口服疫苗可能是超早期调控机体应激、防止脑损伤、减缓药物成瘾和治疗疼痛等的可行方法之一。但需要有载体携带进入血脑屏障发挥作用,而转铁蛋白受体单克隆抗体OX-26是迄今被认为最有前途的脑转运载体,它可携带NR1抗体通过血脑屏障,从而使得NR1抗体能够在中枢神经系统发挥作用。因此,本研究旨在用噬菌体肽库展示技术模拟小鼠NR1和TFR的抗原表位,构建NR1-TfR融合蛋白真核表达载体,构建单B细胞表位疫苗。方法：(1)利用噬菌体十二肽库展示技术筛选小鼠NMDAR1单克隆抗体MAB363的抗原表位筛选(本课题前期研究已完成),筛选小鼠转铁蛋白受体单克隆抗体OX-26的抗原表位优势克隆,(2)计算机模拟表位融合免疫原性蛋白的空间构象,检测其抗原性；(3)将MAB363和OX-26的抗原表位优势克隆通过linker串联人工合成重组表位肽,进行功能检测；(4)将所得目的片段插入真核表达载体pcDNA3.1(+)构建真核表达质粒pcDNA3.1-NR1/TFR,利用电穿孔方法将其转化入减毒伤寒沙门杆菌制备重组表位疫苗；(5)通过western blot技术检测质粒在真核细胞中的表达。结果：(1)通过噬菌体十二肽库展示技术筛选出小鼠转铁蛋白受体单克隆抗体OX-26的抗原表位优势克隆氨基酸序列为GHIHSMRHHRPT.将人工合成的含目的氨基酸DDWVISTQSLKSGGGGSGGGGSGGGGSG HIHSMRHHRPT的多肽分别用MAB363和OX-26进行western blot检测,均能在2KD-8KD范围内产生条带,提示B细胞表位筛选正确,重组表位质粒设计合理,所表达的蛋白能够有效和相应抗体特异性结合。(2)将MAB363和OX-26的抗原表位优势克隆通过linker串联以保证两者的独立功能和空间构象,命名为S。采用相关软件预测重组表位蛋白Pro-S空间构象：Pro-S蛋白质序列在BLAST蛋白数据库中未发现同源性较高(30%)的蛋白质,其内不含信号肽,属非跨膜蛋白,且不含半胱氨酸而未形成二硫化合物影响蛋白折叠和功能发挥,不含α螺旋以保证结构的稳定；NR1和TFR的抗原表位处于蛋白分子的表面,具有良好的抗原性、亲水性,提示Pro-S抗原性较好,可能刺激机体产生免疫反应并产生相应的抗体。(3)成功构建携带目的片段的真核表达质粒pcDNA3.1-NR1/TFR,命名为vec-s;并成功将其转化至减毒沙门菌,制备浓度为1.6×109cfu/ml的减毒活疫苗。(4)将质粒vec-s转染入真核细胞HEK293,48小时候后检测vec-s的瞬时表达,发现vec-s转染后的细胞总蛋白在MAB363和OX-26分别做western blot一抗检测时,在2KD-8KD范围内可见相应条带,与预测的分子量大小符合,提示真核表达质粒vec-s可在真核细胞中表达。结论：成功构建了NR1-TFR重组真核表达质粒,初步验证真核表达质粒vec-s能够在真核细胞中表达目的蛋白。为下一步的动物实验奠定了基础。
[Abstract]:OBJECTIVE: N-methyl-D-aspartate receptor (NR) is widely involved in the pathophysiological processes of stress, brain injury, drug addiction, pain and so on. Selective blockade of NR activity may become a therapeutic tool for related diseases. Existing NR antagonists or blockers are synthetic small molecule drugs because of the choice of action. Low sex often causes serious side effects such as hallucinations, anxiety and blurred consciousness, and it is difficult to use drugs early. NR1 is a functional subunit of NR. It has all the electrophysiological characteristics and pharmacological activities of NR1. One of the most promising brain transporters to date is the monoclonal antibody OX-26, which carries NR1 antibodies across the blood-brain barrier, thus enabling NR1 antibodies to function in the central nervous system. The eukaryotic expression vector of NR1-TfR fusion protein was constructed by mimicking the antigenic epitopes of NR1 and TFR in mice.
METHODS: (1) Screening the epitope of mouse NMDAR1 monoclonal antibody MAB363 by phage display technique, screening the dominant epitope clones of mouse transferrin receptor monoclonal antibody OX-26, (2) Computer simulation of the spatial conformation of epitope fusion immunogenic protein, detecting its anti-NMDAR1 monoclonal antibody MAB363. (3) MAB363 and OX-26 antigen epitope dominant clones were synthesized by linker in series and the recombinant epitope peptide was synthesized for functional detection; (4) The target fragment was inserted into the eukaryotic expression vector pcDNA3.1 (+) to construct the eukaryotic expression plasmid pcDNA3.1-NR1/TFR, which was transformed into attenuated Salmonella typhi by electroporation to prepare the recombinant table. Results: (1) The dominant cloned amino acid sequence of mouse transferrin receptor monoclonal antibody OX-26 was screened out by phage display technique. The synthetic amino acid DDWVISTQSLKSGGGSGGGGSGGGS containing the target amino acid was obtained. The polypeptides of GGGSG HIHSMRHHRPT were detected by Western blot with MAB363 and OX-26, respectively. All the polypeptides could produce bands in the range of 2KD-8KD, suggesting that the B cell epitopes were screened correctly, the recombinant plasmids were designed reasonably, and the expressed proteins could effectively bind to the corresponding antibodies. (2) MAB363 and OX-26 antigen epitope dominant clones were cloned through linker strands. S. Prediction of the spatial conformation of the recombinant epitope protein Pro-S: Pro-S protein sequence was not found to have high homology (30%) in the BLAST protein database, which contained no signal peptide, was a non-transmembrane protein, and did not contain cysteine and did not form disulfide compounds. The antigenic epitopes of NR1 and TFR are located on the surface of protein molecule and have good antigenicity and hydrophilicity, suggesting that Pro-S has good antigenicity and may stimulate the body to produce immune response and corresponding antibodies. (3) Eukaryotic expression of the target fragment was successfully constructed. The plasmid pcDNA3.1-NR1/TFR, named vec-s, was successfully transformed into attenuated Salmonella to prepare live attenuated vaccine with a concentration of 1.6 *109 cfu/ml. (4) Vec-s was transfected into eukaryotic cells HEK293,48 hours later to detect the transient expression of vec-s. The total protein of vec-s transfected cells MAB363 and OX-26 were detected by Western blot, respectively. The corresponding bands were observed in the range of 2KD-8KD, which was in accordance with the predicted molecular weight, suggesting that vec-s could be expressed in eukaryotic cells.
CONCLUSION: The recombinant eukaryotic expression plasmid of NR1-TFR was successfully constructed, which preliminarily verified that vec-s could express the target protein in eukaryotic cells.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392-1

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