Purification And Expression Of Peanut Allergen Ara H1

Peanut is one of the eight most common allergy-causing foods. Generally, peanut allergy is severe, wide spread and typically permanent, which had become a public health issue and food safety problem with an increasing attention. Up to date,11peanut allergens were reported. Among them, Ara h1is an important allergen as recognized by more than90%peanut-allergic individual serum IgE. Therefore, it is necessary and significant to carry out the reseach on Ara h1.The present research is composed of several parts including the purification of Ara h1from nature peanut, preparation of rabbit anti Ara h1polyclonal antibody, cloning and prokaryotic expression of recombinant Ara h1, and purification of the recombinant Ara h1. Major methods and conclusions were listed as following:1. Ara h1was purified from natural peanut seeds through a three-step purification method by three chromatographies:firstly DEAE-Sepharose Fast Flow anion exchange chromatography, then Superdex200gel filtration chromatography and at last ConA Sepharoes4B affinity chromatography. Then the purified Ara h1was identified by SDS-PAGE and MALDI-TOF/MS. The result showed that the purity of Ara h1was above90%and from peanut seed0.43%(W/W) Ara h1could be yielded.2. Specific rabbit polyclonal antibody was raised against Ara h1from New Zealand rabbits, immunizing them with a routine method. The antigenicity of this antibody was evaluated by indirect ELISA, and western blotting. The results showed that the titre of the antibody was1:200,000with high specificity, sufficienting the requirment of future work.3. Total RNA was purified from the peanut seeds using the SV Total RNA Isolation System Kit. Then the DNA fragment of Ara h1was amplified by RT-PCR, and the RT-PCR product was cloned into a pMD18-T-simple Vector. The plasmid was sequenced and analysed. The results showed that the full length cDNA shares99%similarity to reported sequence of Ara h1.4. The target genes were obtained from pMD18-T-Ara h1which was double digested with KpnⅠ and HindⅢ, then cloned into pET-32a expession vector. The recombinant plasmids pET-32a-Ara h1were transformed into Escherichia coli BL21(DE3)plysS. The recombinant proteins were produced with induction by IPTG at37℃and identified by SDS-PAGE and Western bloting. The expression host cell was induced with different concentration of IPTG, different shaking speed, different temperature and different time, respectively. The best conditions of production of soluble recombinant protein were detemined that the IPTG concentration was0.3mM, the shaking speed was160rpm, the induction temperature was24℃and the induction time was eight hours, respectively. The recombint protein was obtained by Ni-NTA metal-affinity chromatography under nondenaturing conditions.