花生过敏原蛋白Ara h1的纯化表达研究

发布时间：2018-06-04 19:37

  本文选题：花生过敏原 + Arah1　； 参考：《南昌大学》2012年硕士论文

【摘要】：花生是世界八大类过敏食物之一,并且由于花生过敏反应具有长期性、普遍性、严重性等特点,已引起全球范围内的广泛关注。现阶段,人们已经发现了11种花生过敏原蛋白,其中Ara h1蛋白含量最高,且被90%的花生过敏患者的血清所识别,是花生主要过敏原之一。因此,本文的主要研究工作围绕Arah1展开。 论文主要工作包括从花生种子中分离纯化天然过敏原Ara h1、兔抗天然Arah1蛋白多克隆抗体的制备、Arah1基因的克隆、重组Arah1的原核表达与纯化。研究的主要方法与结论如下： 1.先后采用DEAE-Sepharose Fast Flow阴离子交换层析、Superdex200凝胶层析及Con A Sepharoes4B亲和层析等三步分离纯化花生过敏原Ara h1,并利用MALDI-TOF/MS对所分离的蛋白进行鉴定。结果表明,本方法可分离纯化得到纯度为90%以上的天然Ara h1,提取得率为43毫克/10克花生。 2.以纯化的天然Ara h1为抗原,免疫新西兰大白兔,获得兔抗Ara h1多克隆抗体,通过ELISA和免疫印迹方法检测其效价和特异性,结果表明,该抗体效价达1：200,000,且不与花生中其他蛋白发生交叉反应,能够满足后续实验的需要。 3.利用SV Total RNA Isolation System试剂盒从花生种子中提取花生总RNA,采用RT-PCR技术扩增获得花生过敏原Ara h1基因,并将其克隆入pMD18-T Simple Vector载体,再导入E. coli DH5a克隆菌保存。对目的基因进行的序列测定表明,该基因与已报道的Arah1序列具有99%的同源性。 4.双酶切重组质粒pMD18-T-Arah1得到Ara h1目的基因,连接至pET-32a表达载体中构建重组质粒pET-32a-Ara h1,并将其转化到表达宿主菌BL21(DE3)plysS中,IPTG诱导表达出重组蛋白Ara h1,并通过SDS-PAGE与免疫印迹实验进行验证。然后对表达宿主菌进行不同IPTG浓度、不同摇床转速、不同诱导温度和时间等条件的优化,获得重组蛋白Ara h1可溶性表达的最佳条件为：IPTG浓度0.3mmol/L,摇床转速160rpm,诱导温度24℃,诱导时间为8h。最后通过亲和纯化的方法获得重组蛋白Ara h1。
[Abstract]:Peanut is one of the eight kinds of allergic food in the world. Because of its long-term, universal and serious characteristics, peanut allergic reaction has attracted worldwide attention. At present, 11 peanut allergen proteins have been found, among which Ara H1 protein is the highest, which is recognized by 90% of peanut allergy patients' serum, and is one of the main allergens in peanut. Therefore, the main work of this paper is focused on Arah1. The main work of this paper is to isolate and purify the natural allergen Ara h1 from peanut seeds, to clone the rabbit polyclonal antibody against natural Arah1 protein, and to express and purify the recombinant Arah1 in prokaryotic. The main methods and conclusions of the study are as follows: 1. DEAE-Sepharose Fast Flow anion exchange chromatography (DEAE-Sepharose Fast Flow) and Con A Sepharoes4B affinity chromatography were used to separate and purify peanut allergen Ara h1. The isolated protein was identified by MALDI-TOF/MS. The results showed that natural Ara H1 with purity of more than 90% could be separated and purified by this method, and the extraction yield was 43 mg / 10 g peanut. 2. The purified natural Ara h1 was used as antigen to immunize New Zealand white rabbits to obtain polyclonal antibodies against Ara h1. The titers and specificity of the polyclonal antibodies against Ara h1 were detected by ELISA and Western blotting. The titer of the antibody was 1: 200000, and the antibody did not cross with other proteins in peanut, which could meet the need of further experiments. 3. Peanut total RNAs were extracted from peanut seeds by SV Total RNA Isolation System kit. The peanut allergen Ara h1 gene was amplified by RT-PCR technique and cloned into pMD18-T Simple Vector vector, and then transferred into E. coli DH5a clone bacteria for preservation. The sequencing of the target gene showed that the gene had 99% homology with the reported Arah1 sequence. 4. The target gene of Ara h1 was obtained by double enzyme digestion of recombinant plasmid pMD18-T-Arah1 and ligated into pET-32a expression vector to construct the recombinant plasmid pET-32a-Ara h1. The recombinant protein Ara h1 was induced by BL21(DE3)plysS and was confirmed by SDS-PAGE and Western blotting. The optimal conditions for soluble expression of recombinant protein Ara H1 were obtained by optimizing the conditions of different IPTG concentration, different shaking speed, different induction temperature and time. The optimal conditions for soluble expression of recombinant protein Ara H1 were obtained as follows: 0.3 mmol / L of Ara concentration, 160 rpm of shaking speed and 24 鈩，

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