腰果主要过敏原Ana o 3的表位谱分析、重组表达及鉴定

发布时间：2018-12-12 05:32

【摘要】：目的:血清特异性IgE是诊断食物过敏的重要指标,已发展至分子诊断水平。抗原表位是抗原与抗体结合的基本单位,通过抗原表位解析,实现特异性IgE的精准检测。本研究以Ana o 3作为研究对象,利用冷休克表达系统获得Ana o 3重组抗原;同时,解析Ana o 3抗原表位,并制备关键抗原表位串联重组蛋白,提高特异性IgE精准检测水平。方法:1.腰果Ana o 3的重组表达:提取腰果总RNA,逆转录至cDNA,设计特异性引物,通过]闶絇CR技术克隆腰果Ana o 3基因,将其插入pCold-SUMO vector,鉴定并测序;重组质粒测序正确后,转化BL21(DE3)感受态细胞,低温15℃诱导表达。摸索诱导表达条件,以便获取可溶性好、表达量高的Ana o 3重组蛋白,镍柱纯化。利用腰果过敏血清作为识别抗体,通过Western blot鉴定免疫活性。2.关键抗原表位的获取:使用三种预测线性抗原表位的软件(DNAsTAR,ABCpred,Bepipred Liner Epitope Prediction)筛选抗原表位。查阅最新关于Ana o3抗原表位研究的文献,并与软件预测结果对比,最终筛选出12条肽段并人工合成。采用斑点印迹技术,以29例腰果Ana o 3过敏患者血清s IgE作为一抗,筛选出反应率高的线性表位作为关键抗原表位。3.关键抗原表位串联重组体的构建及表达:获取关键抗原表位的碱基序列,中间插入柔性肽Linker碱基序列串联,5’端和3’端分别加入StuⅠ和BamHⅠ碱基序列,人工合成抗原表位串联体基因。接着,将该基因与pCold-SUMO质粒进行重组质粒构建,并测序成功。将该重组质粒转入BL21 DE(3)感受态细胞进行重组表达,摸索最优诱导表达条件,镍柱纯化目的蛋白,并进行免疫学活性鉴定。结果:1.从腰果中成功克隆出了Ana o 3基因,并将该基因与pCold-SUMO质粒连接送去测序。将测序结果进行比对分析,发现克隆获取的腰果Ana o 3基因序列为417 bp,与GenBank上Ana o 3基因CDS序列基本一致,而所编码的138个氨基酸则完全相同;SDS-PAGE结果显示,重组Ana o 3分子量为27 kDa左右,大小与理论预测值基本一致;Western blot结果显示,重组Ana o 3与腰果过敏患者血清具有良好的反应性。2.查阅Ana o 3表位的文献,选取12段有反应的肽段作为候选表位。斑点印迹结果显示,12条候选表位肽段与阳性血清呈现不同程度的反应,而11号肽段几乎无反应。统计各候选表位肽段的反应频率,发现1、2、3、6、10号肽段与Ana o 3过敏血清的反应频率几乎均在80%以上,因此将1、2、3、6、10号抗原表位作为关键抗原表位。3.考虑到1、2、3肽段依次有12个氨基酸相互重叠,因此可将其视为同一表位。SDS-PAGE结果显示,抗原表位串联重组蛋白分子量为22 kDa,与理论预测大小相符。Western blot结果显示,抗原表位串联重组蛋白能和Ana o 3过敏血清s IgE呈现良好的反应性。结论:1.基因克隆并于冷休克原核表达系统获得的Ana o 3重组蛋白,表达量高且免疫活性强,证实了该重组Ana o 3作为已知抗原用于腰果过敏sIgE检测的有效性。2.查阅文献及生物信息学预测获得的12个候选抗原表位,除了11号以外,其余均为腰果Ana o 3的抗原表位,预测结果较准确。3.利用冷休克原核表达系统获得的关键抗原表位串联重组蛋白,具有良好的免疫活性,有可能用作腰果Ana o 3血清sIgE检测的优质原料。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.6

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