腰果主要過敏原Ana o 3的表位譜分析、重組表達(dá)及鑒定
發(fā)布時(shí)間：2018-12-12 05:32
【摘要】：目的:血清特異性IgE是診斷食物過敏的重要指標(biāo),已發(fā)展至分子診斷水平�？乖砦皇强乖c抗體結(jié)合的基本單位,通過抗原表位解析,實(shí)現(xiàn)特異性IgE的精準(zhǔn)檢測(cè)。本研究以Ana o 3作為研究對(duì)象,利用冷休克表達(dá)系統(tǒng)獲得Ana o 3重組抗原;同時(shí),解析Ana o 3抗原表位,并制備關(guān)鍵抗原表位串聯(lián)重組蛋白,提高特異性IgE精準(zhǔn)檢測(cè)水平。方法:1.腰果Ana o 3的重組表達(dá):提取腰果總RNA,逆轉(zhuǎn)錄至cDNA,設(shè)計(jì)特異性引物,通過]閌絇CR技術(shù)克隆腰果Ana o 3基因,將其插入pCold-SUMO vector,鑒定并測(cè)序;重組質(zhì)粒測(cè)序正確后,轉(zhuǎn)化BL21(DE3)感受態(tài)細(xì)胞,低溫15℃誘導(dǎo)表達(dá)。摸索誘導(dǎo)表達(dá)條件,以便獲取可溶性好、表達(dá)量高的Ana o 3重組蛋白,鎳柱純化。利用腰果過敏血清作為識(shí)別抗體,通過Western blot鑒定免疫活性。2.關(guān)鍵抗原表位的獲取:使用三種預(yù)測(cè)線性抗原表位的軟件(DNAsTAR,ABCpred,Bepipred Liner Epitope Prediction)篩選抗原表位。查閱最新關(guān)于Ana o3抗原表位研究的文獻(xiàn),并與軟件預(yù)測(cè)結(jié)果對(duì)比,最終篩選出12條肽段并人工合成。采用斑點(diǎn)印跡技術(shù),以29例腰果Ana o 3過敏患者血清s IgE作為一抗,篩選出反應(yīng)率高的線性表位作為關(guān)鍵抗原表位。3.關(guān)鍵抗原表位串聯(lián)重組體的構(gòu)建及表達(dá):獲取關(guān)鍵抗原表位的堿基序列,中間插入柔性肽Linker堿基序列串聯(lián),5’端和3’端分別加入StuⅠ和BamHⅠ堿基序列,人工合成抗原表位串聯(lián)體基因。接著,將該基因與pCold-SUMO質(zhì)粒進(jìn)行重組質(zhì)粒構(gòu)建,并測(cè)序成功。將該重組質(zhì)粒轉(zhuǎn)入BL21 DE(3)感受態(tài)細(xì)胞進(jìn)行重組表達(dá),摸索最優(yōu)誘導(dǎo)表達(dá)條件,鎳柱純化目的蛋白,并進(jìn)行免疫學(xué)活性鑒定。結(jié)果:1.從腰果中成功克隆出了Ana o 3基因,并將該基因與pCold-SUMO質(zhì)粒連接送去測(cè)序。將測(cè)序結(jié)果進(jìn)行比對(duì)分析,發(fā)現(xiàn)克隆獲取的腰果Ana o 3基因序列為417 bp,與GenBank上Ana o 3基因CDS序列基本一致,而所編碼的138個(gè)氨基酸則完全相同;SDS-PAGE結(jié)果顯示,重組Ana o 3分子量為27 kDa左右,大小與理論預(yù)測(cè)值基本一致;Western blot結(jié)果顯示,重組Ana o 3與腰果過敏患者血清具有良好的反應(yīng)性。2.查閱Ana o 3表位的文獻(xiàn),選取12段有反應(yīng)的肽段作為候選表位。斑點(diǎn)印跡結(jié)果顯示,12條候選表位肽段與陽(yáng)性血清呈現(xiàn)不同程度的反應(yīng),而11號(hào)肽段幾乎無(wú)反應(yīng)。統(tǒng)計(jì)各候選表位肽段的反應(yīng)頻率,發(fā)現(xiàn)1、2、3、6、10號(hào)肽段與Ana o 3過敏血清的反應(yīng)頻率幾乎均在80%以上,因此將1、2、3、6、10號(hào)抗原表位作為關(guān)鍵抗原表位。3.考慮到1、2、3肽段依次有12個(gè)氨基酸相互重疊,因此可將其視為同一表位。SDS-PAGE結(jié)果顯示,抗原表位串聯(lián)重組蛋白分子量為22 kDa,與理論預(yù)測(cè)大小相符。Western blot結(jié)果顯示,抗原表位串聯(lián)重組蛋白能和Ana o 3過敏血清s IgE呈現(xiàn)良好的反應(yīng)性。結(jié)論:1.基因克隆并于冷休克原核表達(dá)系統(tǒng)獲得的Ana o 3重組蛋白,表達(dá)量高且免疫活性強(qiáng),證實(shí)了該重組Ana o 3作為已知抗原用于腰果過敏sIgE檢測(cè)的有效性。2.查閱文獻(xiàn)及生物信息學(xué)預(yù)測(cè)獲得的12個(gè)候選抗原表位,除了11號(hào)以外,其余均為腰果Ana o 3的抗原表位,預(yù)測(cè)結(jié)果較準(zhǔn)確。3.利用冷休克原核表達(dá)系統(tǒng)獲得的關(guān)鍵抗原表位串聯(lián)重組蛋白,具有良好的免疫活性,有可能用作腰果Ana o 3血清sIgE檢測(cè)的優(yōu)質(zhì)原料。
[Abstract]:Objective: Serum-specific IgE is an important index in the diagnosis of food allergy and has been developed to the level of molecular diagnosis. the epitope of the antigen is the basic unit of the antigen and the antibody, and the specific IgE can be accurately detected through the analysis of the epitope of the antigen. Ana o-3 recombinant antigen was obtained by using Ana o 3 as a study object and Ana o 3 antigen was obtained by using a cold shock expression system. Method: 1. The recombinant expression of the cashew nut Ana o 3 is as follows: the total RNA of the cashew nut is extracted, reverse transcription is carried out to the cDNA, a specific primer is designed, the cashew nut Ana o 3 gene is cloned by means of the rhodamine CR technology, and the cashew nut Ana o 3 gene is inserted into the pCold-SUMO vector to be identified and sequenced; after the recombinant plasmid is sequenced correctly, the BL21 (DE3) competent cells are transformed, The expression was induced at a low temperature of 15.degree. C. The expression conditions were found to obtain the high-soluble, high-expression Ana o-3 recombinant protein, and the nickel column was purified. The immune activity was identified by Western blot using cashew allergic serum as the identification antibody. The acquisition of the key antigen table bits: the antigen table bits were screened using three software for predicting the linear antigen table bits (DNAsTAR, ABCpred, and Beped Liner Epitope Prediction). The most recent literature on the study of the Ana o3 antigen table was reviewed and compared with the software prediction results, and the 12 peptide fragments were finally screened and artificially synthesized. Using the dot blot technique, the serum s IgE of 29 cashew nut Ana o 3 was used as an anti-tumor, and the linear table position with high reaction rate was selected as the key antigen table. the construction and expression of the key antigen table-position serial recombination body are as follows: the base sequence of the key antigen table position is obtained, the middle inserted flexible peptide Linker base sequence is connected in series, and the 5 'end and the 3' end are respectively added with the Stu I and the BamH I base sequence, and the synthetic antigen table is a tandem gene. Then, the gene was constructed with the pCold-SUMO plasmid and the sequencing was successful. The recombinant plasmid was transferred to a BL21 (DE3) competent cell for recombinant expression, the optimal induction expression condition was found, the protein of the target protein was purified by the nickel column, and the immunological activity was identified. Results: 1. The Ana o 3 gene was successfully cloned from the cashew and the gene was connected to the pCold-SUMO plasmid and sequenced. The result of sequencing was compared with that of Ana o 3 gene in GenBank. The results of SDS-PAGE showed that the molecular weight of recombinant Ana o 3 was about 27 kDa. The results of Western blot show that the recombinant Ana o 3 has good reactivity with cashew allergic patients. Reference the literature of the Ana o 3 table, and select the peptide segment with the reaction in paragraph 12 as the candidate table. The dot blot results showed that the 12 candidate epitope peptide fragments showed different degrees of response to the positive serum, while the 11-peptide fragment was almost non-reactive. The reaction frequency of each candidate epitope peptide segment was counted, and the reaction frequency of the 1, 2, 3, 6, 10 peptide segments and the Ana o 3 sensitive serum was almost 80% or more, and thus the antigen table bits of 1, 2, 3, 6 and 10 were used as the key antigen table bits. In view of the fact that 12 amino acids overlap each other in the 1, 2 and 3 peptide segments, they can be treated as the same table. The SDS-PAGE results show that the molecular weight of the tandem recombinant protein in the epitope is 22 kDa, which is in line with the theoretical prediction. The results of Western blot show that the antigen-epitope tandem recombinant protein can show good reactivity with Ana o-3-sensitive serum s IgE. Conclusion: 1. Ana o 3 recombinant protein obtained by the gene cloning and in the prokaryotic expression system of cold shock has high expression level and strong immune activity, and confirms the effectiveness of the recombinant Ana o 3 as a known antigen for the detection of the cashew allergic sIgE. The 12 candidate antigen table bits obtained from the literature and bioinformatics prediction, except the 11, the rest are the antigen table bits of the cashew Ana o 3, and the prediction results are more accurate. The key antigen epitope tandem recombinant protein obtained by using the cold shock prokaryotic expression system has good immune activity and can be used as a high-quality raw material for detecting the sIgE of cashew Ana o 3.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.6
本文編號(hào)：2374010
[Abstract]:Objective: Serum-specific IgE is an important index in the diagnosis of food allergy and has been developed to the level of molecular diagnosis. the epitope of the antigen is the basic unit of the antigen and the antibody, and the specific IgE can be accurately detected through the analysis of the epitope of the antigen. Ana o-3 recombinant antigen was obtained by using Ana o 3 as a study object and Ana o 3 antigen was obtained by using a cold shock expression system. Method: 1. The recombinant expression of the cashew nut Ana o 3 is as follows: the total RNA of the cashew nut is extracted, reverse transcription is carried out to the cDNA, a specific primer is designed, the cashew nut Ana o 3 gene is cloned by means of the rhodamine CR technology, and the cashew nut Ana o 3 gene is inserted into the pCold-SUMO vector to be identified and sequenced; after the recombinant plasmid is sequenced correctly, the BL21 (DE3) competent cells are transformed, The expression was induced at a low temperature of 15.degree. C. The expression conditions were found to obtain the high-soluble, high-expression Ana o-3 recombinant protein, and the nickel column was purified. The immune activity was identified by Western blot using cashew allergic serum as the identification antibody. The acquisition of the key antigen table bits: the antigen table bits were screened using three software for predicting the linear antigen table bits (DNAsTAR, ABCpred, and Beped Liner Epitope Prediction). The most recent literature on the study of the Ana o3 antigen table was reviewed and compared with the software prediction results, and the 12 peptide fragments were finally screened and artificially synthesized. Using the dot blot technique, the serum s IgE of 29 cashew nut Ana o 3 was used as an anti-tumor, and the linear table position with high reaction rate was selected as the key antigen table. the construction and expression of the key antigen table-position serial recombination body are as follows: the base sequence of the key antigen table position is obtained, the middle inserted flexible peptide Linker base sequence is connected in series, and the 5 'end and the 3' end are respectively added with the Stu I and the BamH I base sequence, and the synthetic antigen table is a tandem gene. Then, the gene was constructed with the pCold-SUMO plasmid and the sequencing was successful. The recombinant plasmid was transferred to a BL21 (DE3) competent cell for recombinant expression, the optimal induction expression condition was found, the protein of the target protein was purified by the nickel column, and the immunological activity was identified. Results: 1. The Ana o 3 gene was successfully cloned from the cashew and the gene was connected to the pCold-SUMO plasmid and sequenced. The result of sequencing was compared with that of Ana o 3 gene in GenBank. The results of SDS-PAGE showed that the molecular weight of recombinant Ana o 3 was about 27 kDa. The results of Western blot show that the recombinant Ana o 3 has good reactivity with cashew allergic patients. Reference the literature of the Ana o 3 table, and select the peptide segment with the reaction in paragraph 12 as the candidate table. The dot blot results showed that the 12 candidate epitope peptide fragments showed different degrees of response to the positive serum, while the 11-peptide fragment was almost non-reactive. The reaction frequency of each candidate epitope peptide segment was counted, and the reaction frequency of the 1, 2, 3, 6, 10 peptide segments and the Ana o 3 sensitive serum was almost 80% or more, and thus the antigen table bits of 1, 2, 3, 6 and 10 were used as the key antigen table bits. In view of the fact that 12 amino acids overlap each other in the 1, 2 and 3 peptide segments, they can be treated as the same table. The SDS-PAGE results show that the molecular weight of the tandem recombinant protein in the epitope is 22 kDa, which is in line with the theoretical prediction. The results of Western blot show that the antigen-epitope tandem recombinant protein can show good reactivity with Ana o-3-sensitive serum s IgE. Conclusion: 1. Ana o 3 recombinant protein obtained by the gene cloning and in the prokaryotic expression system of cold shock has high expression level and strong immune activity, and confirms the effectiveness of the recombinant Ana o 3 as a known antigen for the detection of the cashew allergic sIgE. The 12 candidate antigen table bits obtained from the literature and bioinformatics prediction, except the 11, the rest are the antigen table bits of the cashew Ana o 3, and the prediction results are more accurate. The key antigen epitope tandem recombinant protein obtained by using the cold shock prokaryotic expression system has good immune activity and can be used as a high-quality raw material for detecting the sIgE of cashew Ana o 3.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.6
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