关键词: 花生, 过敏原, Ara h 6, 基因克隆表达, 质谱鉴定

Abstract:

Ara h 6 is one of the major peanut allergens. Ara h 6 cDNA was synthesized from total RNA using Oligo primers
by reverse transcription-polymerase chain reaction (RT-PCR) in order to provide a template for the PCR amplification of
Ara h 6 gene. The target gene was cloned into pMD19-T vector to construct a recombinant vector. Then the recombinant
vector was transferred into the bacterial expression host BL21 (DE3). IPTG was used to induce protein expression, and the
expressed product was purified by Ni affinity chromatography. DNA sequence analysis showed that the full-length gene
fragment of Ara h 6 was 438 bp and encoded 145 amino acids, which was 97% identical to the known DNA sequence.
Sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) results showed that the molecular weight of the
expressed fragment was 24 kD, which matched with the theoretical value of the His-tagged fusion recombinant protein Ara h 6.
Mass spectrometric results showed that the matching degree of structure was 100% between the recombinant protein and
natural Ara h 6. Western blotting indicated that the protein could be recognized by anti-Ara h 6 polyclonal antibody, and has
strong immunogenicity.

Key words: peanut, allergen, Ara h 6, recombinant expression, mass spectrometry