本文選題：花生過(guò)敏原 + Arah1��； 參考：《南昌大學(xué)》2012年碩士論文

【摘要】：花生是世界八大類過(guò)敏食物之一,并且由于花生過(guò)敏反應(yīng)具有長(zhǎng)期性、普遍性、嚴(yán)重性等特點(diǎn),已引起全球范圍內(nèi)的廣泛關(guān)注�，F(xiàn)階段,人們已經(jīng)發(fā)現(xiàn)了11種花生過(guò)敏原蛋白,其中Ara h1蛋白含量最高,且被90%的花生過(guò)敏患者的血清所識(shí)別,是花生主要過(guò)敏原之一。因此,本文的主要研究工作圍繞Arah1展開(kāi)。 論文主要工作包括從花生種子中分離純化天然過(guò)敏原Ara h1、兔抗天然Arah1蛋白多克隆抗體的制備、Arah1基因的克隆、重組Arah1的原核表達(dá)與純化。研究的主要方法與結(jié)論如下： 1.先后采用DEAE-Sepharose Fast Flow陰離子交換層析、Superdex200凝膠層析及Con A Sepharoes4B親和層析等三步分離純化花生過(guò)敏原Ara h1,并利用MALDI-TOF/MS對(duì)所分離的蛋白進(jìn)行鑒定。結(jié)果表明,本方法可分離純化得到純度為90%以上的天然Ara h1,提取得率為43毫克/10克花生。 2.以純化的天然Ara h1為抗原,免疫新西蘭大白兔,獲得兔抗Ara h1多克隆抗體,通過(guò)ELISA和免疫印跡方法檢測(cè)其效價(jià)和特異性,結(jié)果表明,該抗體效價(jià)達(dá)1：200,000,且不與花生中其他蛋白發(fā)生交叉反應(yīng),能夠滿足后續(xù)實(shí)驗(yàn)的需要。 3.利用SV Total RNA Isolation System試劑盒從花生種子中提取花生總RNA,采用RT-PCR技術(shù)擴(kuò)增獲得花生過(guò)敏原Ara h1基因,并將其克隆入pMD18-T Simple Vector載體,再導(dǎo)入E. coli DH5a克隆菌保存。對(duì)目的基因進(jìn)行的序列測(cè)定表明,該基因與已報(bào)道的Arah1序列具有99%的同源性。 4.雙酶切重組質(zhì)粒pMD18-T-Arah1得到Ara h1目的基因,連接至pET-32a表達(dá)載體中構(gòu)建重組質(zhì)粒pET-32a-Ara h1,并將其轉(zhuǎn)化到表達(dá)宿主菌BL21(DE3)plysS中,IPTG誘導(dǎo)表達(dá)出重組蛋白Ara h1,并通過(guò)SDS-PAGE與免疫印跡實(shí)驗(yàn)進(jìn)行驗(yàn)證。然后對(duì)表達(dá)宿主菌進(jìn)行不同IPTG濃度、不同搖床轉(zhuǎn)速、不同誘導(dǎo)溫度和時(shí)間等條件的優(yōu)化,獲得重組蛋白Ara h1可溶性表達(dá)的最佳條件為：IPTG濃度0.3mmol/L,搖床轉(zhuǎn)速160rpm,誘導(dǎo)溫度24℃,誘導(dǎo)時(shí)間為8h。最后通過(guò)親和純化的方法獲得重組蛋白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，

本文編號(hào)：1978592