Prokaryotic Expression, Purification And Immune Activity Analysis Of 16KDa Major Allergen From Tartary Buckwheat And Transformation Of Arabidopsis Thaliana

Tartary buckwheat has a high nutritional value and medicinal value, its protein, fat and fiber are generally higher than that of other food crops, and it contains chlorophyll and biological flavonoids which were not found in other crops. However, the allergic components of tartary buckwheat hinder the development and utilization of tartary buckwheat food. In recent years, allergic components of common buckwheat had been researched by many scholars at home and abroad in a wide range, in which common buckwheat 16kDa allergens was reported more.The common buckwheat 16kDa allergen, as a 2S albumin family, is considered the major common buckwheat allergen with strong pepsin-resistance activity. Also it was reported that common buckwheat 16kDa allergen was associated with anaphylactic shock. With regard to the allergic composition in tartary buckwheat, less relevant reports at home and abroad has been found, now mostly identified allergens are high molecular weight allergen protein.In this study the nucleic acid sequence analysis and functional structure prediction were performed using the EST sequences(GenBank accession number: GO496294) isolated from the seed-filling periods cDNA library of tartary buckwheat, and the results showed that the gene contains 450bp open reading frame (ORF) which encoded 149 amino acid residues with molecular weight of 17.2kDa, including a putative signal peptide. Homologous analysis showed that the amino acid sequence was 83% homologous to the reported buckwheat 16kDa allergen. Tertiary structure and secondary structure prediction results showed that the encoded protein was a compact structure containing fourα-helix structure. The functional structure prediction showed it included anα-amylase inhibitor (AAI) domain. Six possible linear epitopes were found by antigen epitope perdiction.The full-length genes(450bp / 149aa) and part of the gene(384bp/23-149aa)without the signal peptide were used for the structure of prokaryotic expression vector pET47b-TBW16 and pET47b-cTBW16, respectively, which were transformed into E. coli for prokaryotic expression. It was found that two protein (rTBW16 and rcTBW16) were expressed in the form of inclusion bodies in E. coli Rosetta gami 2(DE3) and BL21 star(DE3), respectively. Both pure recombinant protein were obtained by using inclusion body purification, renaturation and cobalt affinity chromatography, respectively. Comparing with rcTBW16, a large number of sediment of rTBW16 was observed, which has a low efficiency of renaturation in the course of inclusion bodies refolding. Competitive ELSIA was carried out using ncTBW16 and rcTBW16 for immunization activity detection respectively, and found that both ncTBW16 and rcTBW16 had a high immune activity. IgE immunoblot analysis of total protein of tartary buckwheat seeds and rcTBW16 were carried out using two serum from buckwheat allergic patients and two control serum, and result showed that the hybridization signal of two positive serum was noted in 16kDa, which show that the protein band is the 16kDa allergen in tartary buckwheat. This allergen has been named Fag t 2 by IUIS(International Union of Immunological Societies) Allergen Nomenclature Sub-committee.High titer polyclonal antibodies were producted using rcTBW16. Western Blotting analysis of tartary buckwheat seed total protein, albumin, globulin and HCl extracted protein showed that tartary buckwheat 16kDa allergens was in the albumin, which indicated that it is a member of 2S albumin family. In addition, Western Blotting analysis of tartary buckwheat seed embryo and endosperm showed that tartary buckwheat 16kDa allergens exist only in embryo.ncTBW16 was isolated and identified by using technologies such as PAGE , Western Blotting, gel recycling, the N-terminal amino acid sequencing and mass spectrometry. The results showed that the measured N-terminal amino acid sequence was identical to that of the mature and native common buckwheat 16kDa allergen, and mass spectrometry results also show that the isolated tartary buckwheat 16kDa protein corresponds exactly to rcTBW16.In the neutral condition thermal stability analysis of rcTBW16 and ncTBW16 was performed by using SDS-PAGE and ELSIA technology, the results showed that both were in the supernatant by centrifugation after 150min heating at 100 degrees, and there are still 94% of IgE binding activity exists. Pepsin digestion test was carried out based on Satoru's method using ncTBW16 and rcTBW16, and the result showed that both were resistant to pepsin. In addition, the method of 3,5 - dinitrosalicylic acid was carried out to verify theα-amylase inhibitor activity of rcTBW16, and the result showed rcTBW16 did not inhibit the activity ofα-amylase.The plant expression vector pBIN438-TBW16 was constructed and transformed into Arabidopsis by Agrobacterium-mediated. The positive plant was obtained via the screening of kanamycin antibiotics.