Ara h 2. pet - 32a + Origami. Western blotting ELISA SDS - PAGE Ara h 2 F - Ara h 2. IgE

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1 http / /xuebao. jxau. edu. cn Acta Agriculturae Universitatis Jiangxiensis E - mail ndxb7775@ sina. com Ara h * 2 4* Ara h 2 Ara h 2 pet - 32a + Origami Isopropyl β - D Thiogalactopyranoside IPTG Ni2 + Western blotting ELISA pet - 32a + SDS - PAGE Western blotting ELISA Ara h 2 F - Ara h 2 Ara h 2 R - Ara h 2 IgE Ara h 2 Ara h 2 Q786 A Preparation and Characterization of the Genetically Engineered Hypoallergenic Derivative of the Major Peanut Allergen Ara h 2 YI Hai-tao 1 2 LIU Fang 3 JIA Meng-yang 4 TANG Mu-jing 5 CAO Xiao-yong 6 XIA Li-xin 2 4* LIU Zhi-gang 2 4* 1. College of Life Science Shenzhen University Shenzhen China 2. Institute of Allergy and Immunology Shenzhen University Shenzhen China 3. Xinjiang Huashidan Pharmaceutical Co. Ltd Wulumuqi China 4. Medical College Shenzhen University Shenzhen China 5. Shenzhen Entry - Exit Inspection and Quarantine Bureau Shenzhen China 6. Maternal and Child Health Hospital of Futian Shenzhen China Abstract The genetically engineered major allergen Ara h 2 was expressed and purified and the hypoallergenic of purified recombinant F - Ara h 2 protein was characterized. The Ara h 2 was reassembled and inserted into the expression vector pet - 32a +. The vector was transformed into Origami and the protein expression was induced by IPTG. Ni 2 + Chelating affinity chromatography was used to purify the recombinant F SZ E - mail yihaitao1022@ 163. com * E - mail lzg@ szu. edu. cn E - mail xialixin@ 126. com

2 Ara h 2 protein. The hypoallergenic of F - Ara h 2 was examined by Western blotting and ELISA. The ORF which contained 471 bp and encoded 157 amino acids was authenticated to be F - Ara h 2. The recombinant F - Ara h 2 protein induced by IPTG was consistent with the actual value. The affinity between recombinant F - Ara h 2 protein and IgE antibodies from pooled peanut - allergic patients serum was significantly decreased compared with R - Ara h 2 was identified by Western blotting and ELISA. Recombinant F - Ara h 2 protein was hypoallergenic. Key words genetic engineering peanut allergen Ara h 2 hypoallergenic % % WHO SIT Ara h 2 IgE F - Ara h E. coli Top10 Origami Gel Extraction Kit 50 D Plasmid Mini KitⅠ 100 D OMEGA EcoRⅠ HindⅢ T4 DNA pet - 32a + DL2 000 DNA Marker TaKaRa Marker SM0671 Fermentas MBI IgE HRP IgE KPL DAB Invitrogen Ara h 2 3 IgE EcoRⅠ gaattc HindⅢ aagctt g ggg puc57 simple vector F - Ara h 2 F - Ara h 2 puc57 simple vector EcoRⅠ HindⅢ F - Ara h pet - 32a + F - Ara h 2 puc57 simple vector F - Ara h 2 F - Ara h 2 pet - 32a + EcoRⅠ HindⅢ T4DNA E. coli Top pet - 32a + F - Ara h 2 Origami pet - 32a + F - Ara h 2 Origami IPTG 20 mmol /LTris - HCl ph 8. 0 SDS - PAGE F - Ara h 2 SDS - PAGE Ni mmol /L 100 mmol /L 300 mmol /L

3 5 Ara h SDS - PAGE Western blotting F - Ara h 2 Marker R - Ara h 2 F - Ara h 2 SDS - PAGE F - Ara h 2 R - Ara h 2 2 μg 300 ma SDS - PAGE 8 HRP IgE DAB ELISA F - Ara h 2 A1 - D ng F - Ara h μl A4 - D6 200 ng F - Ara h 2 A7 - D9 400 ng F - Ara h 2 A9 - D ng F - Ara h 2 96 E1 - H3 100 ng R - Ara h μl E4 - H6 200 ng R - Ara h 2 E7 - H9 400 ng R - Ara h 2 E9 - H ng R - Ara h PBST 3 5 min / 100 μl2% PBST - BSA37 2 h IgE h PBST 3 5 min / Ara h 2 Original Ara h 2 F - Ara h 2 = = IgE IgE 1 Ara h 2 Original represents the original amino acid sequence of Ara h 2 F - Ara h 2 represents the amino acid sequence of Ara h 2 which is reformed by genetic engineering = = represents 1 h the major IgE epitope of Ara h 2. 1 F - Ara h 2 R - Ara h 2 Fig. 1 Amino acid sequence alignment between F - Ara h 2 and R - Ara h h TMB min 2 mol /L 450 nm SPSS 4 F - Ara h 2 R - Ara h Ara h 2 F - Ara h 2 Ara h 2 BioEdit F - Ara h 2 R - Ara h puc57 simple vector F - Ara h 2 2 puc57 simple vector F - Ara h 2 EcoRⅠ HindⅢ F - Ara h pet - 32a + F - Ara h 2 EcoRⅠ THindⅢ pet - 32a + F - Ara h bp pet - 32a + F - Ara h 2 SDS - PAGE pet - 32a + F - Ara h 2 Origami

4 M DNA Marker DL puc57 simple vector F - Ara h 2 M DNA Marker DL Recombinant plasmid puc57 simple vector - F - Ara h 2 digested by restriction enzymes. 2 puc57 simple vector - F - Ara h 2 EcoRⅠ HindⅢ Fig. 2 puc57 simple vector - F - Ara h 2 digested by restriction enzymes EcoRⅠand HindⅢ M DNA Marker DL pet - 32a + F - Ara h 2 M DNA Marker DL Recombinant plasmid pet - 32a + - F - Ara h 2 digested by restriction enzymes. 3 pet - 32a + F - Ara h 2 EcoRⅠ HindⅢ Fig. 3 Restriction enzymes digestion analysis of recombinant plasmid pet - 32a + - F - Ara h 2 SDS - PAGE 40 mmol /L 100 mmol /L 300 mmol /L F - Ara h mmol /L Marker 300 mmol /L SDS - PAGE kda pet - 32a + N 6 His - Tag 20 kda F - Ara h 2 17 kda 2. 5 Western blotting F - Ara h 2 Western blotting R - Ara h 2 37kDa F - Ara h 2 37 kda R - Ara h 2 ELISA R - Fig. 4 Ara h 2 F - Ara h 2 M Marker 1 pet - 32a + F - Ara h 2 Origami F - Ara h R - Ara h 2 5 F - Ara h 2 IgE M Protein marker 1 E. coli Origami /pet -32a - F - Ara h 2 before induction ELISA F - Ara h 2 2 E. coli Origami /pet - 32a + - F - Ara h 2 after induction by IPTG for 4 h 3 The supernatant of ultrasound 4 The precipitation of ultrasound 5 The recombinant F - Ara h 2 purified by affinity chromatography. 4 F - Ara h 2 Expression and purification of recombinant F - Ara h 2 protein

5 5 Ara h SPSS P < R - Ara h 2 F - Ara h 2 F - Ara h 2 IgE R - Ara h M Marker 1 Ara h 2 R - Ara h 2 2 Ara h 2 F - Ara h 2 IgE I M Protein marker 1 R - Ara h 2 2 F - Ara h F - Ara h Fig. 5 Western blotting analysis of recombinant F - Ara h 2 protein R - Ara h 2 Ara h 2 F - Ara h 2 Ara h 2 *** P < R - Ara h 2 F - Ara h R - Ara h 2 represents recombinant Ara h 2 protein F - Ara h 2 represents genetically engineered Ara h 2 protein *** P < represents the difference between R - Ara h 2 and F - Ara h 2 is Extremely significant. 6 F - Ara h 2 ELISA Ara h 2 Fig. 6 ELISA analysis of recombinant F - Ara h 2 protein IgE T Ara h 2 3 IgE T 3 IgE Western - blotting ELISA F - Ara h 2 IgE R - Ara h 2 R - Ara h 2

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