NR1-TFR重組表位疫苗的制備及其在真核細(xì)胞中的表達(dá)
發(fā)布時間：2018-09-12 13:22
【摘要】：目的：N—甲基—D—天冬氨酸受體(N-Methyl-D-Asparate Receptor, NR)廣泛參與應(yīng)激、腦損傷、藥物成癮、疼痛等病理生理過程。選擇性阻斷NR活性可能成為相關(guān)疾病的治療手段�，F(xiàn)有的NR拮抗劑或阻斷劑均為人工合成的小分子藥物,由于作用選擇性低常引起幻覺、焦慮不安、意識模糊等嚴(yán)重毒副作用,并存在難以超早期用藥的問題。NR1是NR的功能亞單位,具有NR1的一切電生理學(xué)特性和藥理學(xué)活性。NR1口服疫苗可能是超早期調(diào)控機體應(yīng)激、防止腦損傷、減緩藥物成癮和治療疼痛等的可行方法之一。但需要有載體攜帶進(jìn)入血腦屏障發(fā)揮作用,而轉(zhuǎn)鐵蛋白受體單克隆抗體OX-26是迄今被認(rèn)為最有前途的腦轉(zhuǎn)運載體,它可攜帶NR1抗體通過血腦屏障,從而使得NR1抗體能夠在中樞神經(jīng)系統(tǒng)發(fā)揮作用。因此,本研究旨在用噬菌體肽庫展示技術(shù)模擬小鼠NR1和TFR的抗原表位,構(gòu)建NR1-TfR融合蛋白真核表達(dá)載體,構(gòu)建單B細(xì)胞表位疫苗。 方法：(1)利用噬菌體十二肽庫展示技術(shù)篩選小鼠NMDAR1單克隆抗體MAB363的抗原表位篩選(本課題前期研究已完成),篩選小鼠轉(zhuǎn)鐵蛋白受體單克隆抗體OX-26的抗原表位優(yōu)勢克隆,(2)計算機模擬表位融合免疫原性蛋白的空間構(gòu)象,檢測其抗原性；(3)將MAB363和OX-26的抗原表位優(yōu)勢克隆通過linker串聯(lián)人工合成重組表位肽,進(jìn)行功能檢測；(4)將所得目的片段插入真核表達(dá)載體pcDNA3.1(+)構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.1-NR1/TFR,利用電穿孔方法將其轉(zhuǎn)化入減毒傷寒沙門桿菌制備重組表位疫苗；(5)通過western blot技術(shù)檢測質(zhì)粒在真核細(xì)胞中的表達(dá)。結(jié)果：(1)通過噬菌體十二肽庫展示技術(shù)篩選出小鼠轉(zhuǎn)鐵蛋白受體單克隆抗體OX-26的抗原表位優(yōu)勢克隆氨基酸序列為GHIHSMRHHRPT.將人工合成的含目的氨基酸DDWVISTQSLKSGGGGSGGGGSGGGGSG HIHSMRHHRPT的多肽分別用MAB363和OX-26進(jìn)行western blot檢測,均能在2KD-8KD范圍內(nèi)產(chǎn)生條帶,提示B細(xì)胞表位篩選正確,重組表位質(zhì)粒設(shè)計合理,所表達(dá)的蛋白能夠有效和相應(yīng)抗體特異性結(jié)合。(2)將MAB363和OX-26的抗原表位優(yōu)勢克隆通過linker串聯(lián)以保證兩者的獨立功能和空間構(gòu)象,命名為S。采用相關(guān)軟件預(yù)測重組表位蛋白Pro-S空間構(gòu)象：Pro-S蛋白質(zhì)序列在BLAST蛋白數(shù)據(jù)庫中未發(fā)現(xiàn)同源性較高(30%)的蛋白質(zhì),其內(nèi)不含信號肽,屬非跨膜蛋白,且不含半胱氨酸而未形成二硫化合物影響蛋白折疊和功能發(fā)揮,不含α螺旋以保證結(jié)構(gòu)的穩(wěn)定；NR1和TFR的抗原表位處于蛋白分子的表面,具有良好的抗原性、親水性,提示Pro-S抗原性較好,可能刺激機體產(chǎn)生免疫反應(yīng)并產(chǎn)生相應(yīng)的抗體。(3)成功構(gòu)建攜帶目的片段的真核表達(dá)質(zhì)粒pcDNA3.1-NR1/TFR,命名為vec-s;并成功將其轉(zhuǎn)化至減毒沙門菌,制備濃度為1.6×109cfu/ml的減毒活疫苗。(4)將質(zhì)粒vec-s轉(zhuǎn)染入真核細(xì)胞HEK293,48小時候后檢測vec-s的瞬時表達(dá),發(fā)現(xiàn)vec-s轉(zhuǎn)染后的細(xì)胞總蛋白在MAB363和OX-26分別做western blot一抗檢測時,在2KD-8KD范圍內(nèi)可見相應(yīng)條帶,與預(yù)測的分子量大小符合,提示真核表達(dá)質(zhì)粒vec-s可在真核細(xì)胞中表達(dá)。 結(jié)論：成功構(gòu)建了NR1-TFR重組真核表達(dá)質(zhì)粒,初步驗證真核表達(dá)質(zhì)粒vec-s能夠在真核細(xì)胞中表達(dá)目的蛋白。為下一步的動物實驗奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392-1
本文編號：2239132
[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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392-1
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