| Buckwheat is a kind of valuable plant with great advantages in nutrition and medicine. Its high content of bioactive substances has showed significant effects in preventing currently common cardiovascular diseases such as diabetes, hypertention etc. and some other diseases including rheumatic arthritis. However, another unfavorable reaction, buckwheat allergy, which was aroused by touching or eating buckwheat food, has brought people much trouble. Many scientists have embarked on the research of the reasonable allergen in common buckwheat (CB). Considering comparatively few research at present on the allergen in tartary buckwheat (TB), it is necessary for some attention and concentration in this field.Till now, a protein with 22kDa in tartary buckwheat (TB22kDa) has been identified as the major allergen, so further study of the corresponding epitope in virtue of immunological and molecular genetic methods will be helpful in understanding the molecular mechanism of allergenic reaction and finding proper ways of resisting irritability. We paid our main attention to cloning TB22kDa gene from tartary buckwheat, expressing the structure gene and getting the purified recombinant protein, and these provide foundation for father study on the relationship between structure and function of the allergenic protein TB22kDa, the orientation of the corresponding epitope and the exploration of allergenic reaction mechanism.According to the amino acid sequence resulted from our previous research about TB22kDa protein and the related literatures about gene sequence of allergenic protein in common buckwheat, we designed primers and got the structure gene successfully. By 3'-RACE method combined with nested PCR, the 3'-end nuclear acid sequence was also obtained; In additon, for the 5'-end sequence, we selected a specific conserved nuclear acid sequence as the 5'primer and part of structure gene sequence as the 3' primer, and till now, partial 5'-end sequence has been amplified as well. The gene fragment we have got is 1311bp long, which contains a length of 543bp from 5'-end to the coding region, 588bp of open reading frame including" the coding region of 585bp and a termination codon as well as the 3'-end sequence of 180bp rich in AT (accounting for 70%).Gene sequence analysis showed that the fragment we have obtained has 94% and 90% homology with common buckwheat storage protein and legumins, respectively. Meanwhile, by analyzing the amino acid sequence, it shares 93%-83% homology with legumins from common buckwheat and sword bean. Compared relative similarity with the amino acid sequence of reported allergen protein, a peptide fragment located at the site of 183-188, KEEEKE, was common to different allergens, so we speculated that it might act as the potential epitope.Now, the structure gene sequence and 3'-end gene sequence have been logged on GenBank. The accession number is AY044918. This is the first report worldwide of the allergenic protein gene sequence from tartary buckwheat.The amplified PCR product of coding region of TB22kDa was ligated into expression vector pQE-31 and the recombinant plasmid was transformed into E.coli M15 strain. The geneticallyengineered strain was incubated at 37 until OD600 reached at 0.5-0.7, then IPTG was added to final concentration of 0.5 mmol/L to induce the expression of TB22kDa gene. The analysis of SDS-PAGE indicated a fusion protein band at the site of 20-30kDa appeared in the form of inclusion body. The purity of expressed protein finally reached to about 90% after affinity chromatography with HiTrap?Chelating column following the denaturalization treatment.
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