Vol. 39 No Journal of Jiangxi Normal University Natural Science Jan Western blot %. 40 kda

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1 39 1 Vol 39 No Journal of Jiangxi Normal University Natural Science Jan * R A DOI /j cnki issn pet-28a E coli Top10 -β- D- IPTG Western Blot ELISA - SDS-PAGE 40 kda Western blot α 16 38% 18 93% 64 69% Western Blot ELISA I % pet-28a Eco R I Bam H I TaKa- 4 Ra Horseradish Peroxidase HRP lge Southern Biotech BL21 DE3 Chapman Ni-NAT martrix GE Amersham Der p1 Pharmacia Biotech BioTek Ⅰ gjhz0009 GJHZ JCYJ JCYJ KC2012JSYB0003A 1986-

102 2015 1 μg L - 1 100 μl 96 37 2 h 5% PBS 4 1 2 3% PBST 100 μl 3% 1 2 1 PBST HRP IgE TMB A B = 1 1 37 10 min 50 μl 2 mol L - 1 H 2 SO 4 OD 450 1 2 5 pmd18-t E coli Top10 Amp Amp BioEdit ProtParam

2 μg L μl h 5% PBS % PBST 100 μl 3% PBST HRP IgE TMB A B = min 50 μl 2 mol L - 1 H 2 SO 4 OD pmd18-t E coli Top10 Amp Amp BioEdit ProtParam LB DNA Eco R I Xho I ABCpred Prediction MEGA5 1 Clustalx Eco R I Xho I 2 pet-28a pmd18-t- 2 1 pet-28a pet-28a bp E coli Top NCBI http / /www ncbi nlm nih gov /gorf /gorf html pet-28a r min IPTG 1 0 mol L r min h 1 ml 4 1 PBS 100 μl 20 μl 5 loading buffer 10 min 10 μl 10% SDS-PAGE pet Ni + 1 Ni pet-28a Western pet-28a Eco R I Blot SDS-PAGE Xho I SDS-PAGE PVDF cdna bp 5% TBS 37 2 h 2 5% TBST IgE HRP 2 3 ECL Ni + SDS-PAGE 2 ELISA 40 ku

1 103 3-80 M DNA 1 pet28a- 2 pet28a- M 1 2 4 Western Blot 2 5 1 M 1 2 3 ProtParam 39 374 0 Da 6 12 Instability 3 index 36 99 2 4 69 75 Grand

Cpred Prediction IgE 2 4 2 ELISA 4 36 ~ 51 44 ~ 59 81 ~ 96 107 ~ 122 HRP IgE TMB 131 ~ 146 ~ 161 189 ~ 204 234 ~ 249 257 ~ 272 H 2 SO 4 OD 450 5

3 M DNA 1 pet28a- 2 pet28a- M Western Blot M ProtParam Da 6 12 Instability 3 index Grand average of hydropathicity TMHMM Western Blot SDS-PAGE PVDF 20 Western Blot ECL 4 40 ku HNN α 16 38% β 18 93% 64 69% AB- Cpred Prediction IgE ELISA 4 36 ~ ~ ~ ~ 122 HRP IgE TMB 131 ~ 146 ~ ~ ~ ~ 272 H 2 SO 4 OD OD NCBI IgE Blastp Blomia tropicalis Ixodes scapularis Meta- seiulus occidentalis Saccoglossus kowa- 5 ELISA ~ ~ ~ ~ 320

104 2015 levskii Pediculus humanus corporis MEGA5 1 Clustalx1 83 Tribolium castaneum Crassostrea 7 gigas Danaus plexippus AAQ24554 1 XP - Dendroctonus

4 levskii Pediculus humanus corporis MEGA5 1 Clustalx1 83 Tribolium castaneum Crassostrea 7 gigas Danaus plexippus AAQ XP - Dendroctonus ponderosae Daphnia pulex Sus scrofa Fasta Western Blot 11 ~ 100 kda 7 ELISA Westernblot Der f2 IgE 24 8 Der f1 α kd 9-10

5 Eric Weber Ph D Shirley Hunter Ph D Kim Stedman B S et al Identification characterization and cloning of a complementary DNA encoding a 60-kd house dust mite allergen Der f 18 for human beings and dogs J Jallergy 1 Clin Immunol J 9Seung Youp Shin Gyu Young Hur Young Min Ye et al A Masashi Suzuki Yoshimasa Tanaka Seigo Korematsu et al Crystal structure and some properties of a major house dust mite allergen Derf 2 J Biochemical and Biophysical Research Communications Wayne R Thomas Belinda J HalesWendy-Anne Smith House dust mite allergens in asthma and allergy J Trends in Molecular Medicine Susanne Vrtala HansHuberWayne R Thomas Recombinant house dust mite allergens J Methods Der p2 J Der f15 J case of occupational rhinitis caused by porcine pancreatic extract developing into occupational asthma J J Korean Med Sci Brant Hole Cannon et al Occupational asthma caused by cellulase and lipase in the detergent industry J Occup Environ Med Yu Hui Ling Li Jun Qian et al Efficacy analysis of threeyear subcutaneous SQ-standardized specific immunotherapy in house dust miteallergic children with asthma J Experimental and Therapeutic Medicine Kyoung Yong Jeong Jae-Hyun Lee Eun-Jin Kim et al Current status of standardization of inhalant allergen extracts in korea J Allergy Asthma Immunol Res Bozek A Ignasiak B Filipowska B et al House dust mite sublingual immunotherapy a double-blindplacebo-con trolled study in elderly patients with allergic rhinitis J J Clinical & Experimental Allergy Prokaryotic Expression Purification and Bioinformatics of Lipase Arecombinant Allergen of Dust Mite XING Peng 1 LIU Yulin 2 YU Haiqiong 1 LI Meng 1 LIU Zhigang LIU Xiaoyu 1* 1 Allergy and Immunology Institute of Shenzhen University Shenzhen Guangdong China 2 Immunology Department of Nanchang University Medical School Nanchang Jiangxi china Abstract To clone express and purify of dust mite lipase test its immunogenicity and analysis of its structure and function Synthesize the lipase gene of dust mite link it to the pet-28a vector and the recombinant plasmid was transfected into E coli Top10 and IPTG induces the expression of lipase protein The protein was purified by nickelaffinity chromatography and its immunological identification was analyzed by Western blot and ELISA Bioinformatics software were used to predict physicochemical properties secondary structure and to construct its molecular phylogenetic tree High purify recombinant lipase protein of dust mite was successful expressed purified SDS-PAGE results showed that the expression of recombinant allergen was about 40 kda After chromatography the recombinant allergen could band with the serum IgE from patients with asthma in Western Blot Its secondary structure contained an alpha helix 16 38% extended strand 18 93% and random coil 64 69% The success of prokaryotic expression and of dust mite allergen lipase and purified with high purity and strong immunological activity of the recombinant lipase protein It can lay the foundation for the special diagnosis of allergic diseases Key words dust mites allergen recombination lipase bioinformatics analysis

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