Cloning, Recombinant Protein Expression of Rice 14-16 kDa Allergens and Purification, Characterization and Detection of Allergenic Proteins

• Main Authors: Shu-Ling Huang, 黃淑玲
• Other Authors: 黃文哲
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/80523112500853072533

博士 === 國立中興大學 === 食品暨應用生物科技學系所 === 99 === Rice is the staple food in Asia, it plays an important role in providing energy to general population within the region. Rice 14-16 kDa allergenic proteins, a salt-soluble protein fraction, were the products of homologous genes with similar structures. The 14-16 kDa allergenic proteins have been reported as human salivary α-amylase inhibitor, barley trypsin inhibitor and storage protein of grate asako. It was demonstrated that these proteins were the major allergens which caused allergic reactions with consumption of cereals. It was reported that the 14-16, 26, 33 and 56 kDa proteins in rice could react with IgE of rice allergic patients in which the 14-16 kDa proteins, especially, were found to be recognized by IgE antibodies from 90-95% of rice allergic patients. Therefore, 14-16 kDa allergens are thought to be the main allergy proteins in rice. Environmental pollution and dietary factors on human allergenicity has drawn much attention lately. More information in this regard is needed. To develop an allergen-specific antibody is the first step for food allergen detection. It is difficult to obtain the specific antibodies against rice 14-16 kDa allergens. The objectives of this study were to utilize the molecular transgenic technology to amplify rice 14-16 kDa allergen genes and transfer the genes into the expression vector followed by expressing the target recombinant protein in Escherichia coli expression host, to purify target recombinant protein, and to study the use of purified protein on detection of rice 14-16 kDa allergens. pET-29a(+)-RA17 Expression vector of rice was constructed by using the RT-PCR method to amplify the cDNA of RA17 (one of rice 14-16 kDa allergens) from rice mRNA. Recombinant RA17 (rRA17) was expressed in Escherichia coli expression host as a histidine-tag fusion protein and was purified to its homogeneity. The polyclonal antibody was then prepared from the rRA17 protein to be further used as a tool to analyze the content of 14-16 kDa allergens in phytase-transgenic rice (GR) and non-transgenic rice (NR) (wild-type, Oryza sative L. cv. Tainung 67). Result of western blotting showed that the content of major allergenic proteins in GR was 96.3% of that in NR, which were 1.66 ± 0.08 mg/g and 1.73 ± 0.09 mg/g for GR and NR, respectively. It appeared that transferring phytase gene into rice plant did not affect the content of rice major allergenic proteins significantly. The 14-16 kDa allergens of rice was purified and was further analyzed and characterized. Salt-souble proteins of rice were first extracted with 1M NaCl solution followed by precipitation with 90% saturated ammonium sulfate solution. The precipitate was dialyzed and then subjected to DEAE-Sephadex ion-exchange chromatography. Four fractions (I-IV) were obtained. Each fraction was further purified by Sephadex G-50 gel-filtration. The purified fractions were identified as RA14/RA14B (FDEAE-Peak I), RA14C/RAG2 (FDEAE-Peak II), similar to RA17 precursor (FDEAE-Peak III ) and RA14/RA14B (FDEAE-Peak IV) by LC-MS-MS analysis. In a simulated gastric fluid (SGF) test, both RA14/RA14B and RA14C/RAG2 were fully digested in 5 min whereas similar to RA17 precursor was stable up to 60 min. In contrast, both RA14/RA14B and RA14C/RAG2 were relatively stable when subjected to a simulated intestinal fluid (SIF) test. Besides, RA14/RA14B, RA14C/RAG2 and similar to RA17 precursor remained to be stable when heated at 100°C for 60 min. Contents of 14-16 kDa allergens in 9 studied rice samples were as follows: 2.36 ± 0.12 mg/g (Oryza sative L. cv. Taikeng 2), 2.16 ± 0.13 mg/g (Oryza sative L. cv. Taikeng 4), 1.91 ± 0.11 mg/g (Oryza sative L. cv. Taikeng 9), 2.26 ± 0.10 mg/g (Oryza sative L. cv. Taoyuan 3), 2.61 ± 0.07 mg/g (Oryza sative L. cv. Tainan 11), 1.81 ± 0.11 mg/g (Oryza sative L. cv. Kaohsiung 139), 2.26 ± 0.09 mg/g (Oryza sative L. cv. Kaohsiung 145), 2.12 ± 0.10 mg/g (Oryza sative L. cv. Taisen 22), 1.95 ± 0.09 mg/g (Imported Thailand Indica rice), 1.69 ± 0.08 mg/g (Sihu long waxy rice), 1.67 ± 0.12 mg/g (Xinhua long waxy rice), 1.73 ± 0.11 mg/g (Sihu round waxy rice) and 1.80 ± 0.10 mg/g (Siluo round waxy rice). Data indicated that there was no correlation between contents of 14-16 kDa allergens and growing areas of rices. Furthermore, the 14-16 kDa allergen contents were different in some japonica and indica rice strains. In SGF test, the 14-16 kDa allergens in tested rices could be completely digested in 2 min, and all fragments after SGF digesting showed no allergic activity when subjected to Western blotting.