39 1 Vol 39 No 1 2015 1 Journal of Jiangxi Normal University Natural Science Jan 2015 1000-5862 2015 01-0101-05 1 2 1 1 1 1* 1 518060 2 330006 R 392 11 A DOI 10 16357 /j cnki issn1000-5862 2015 01 19 pet-28a E coli Top10 -β- D- IPTG Western Blot ELISA - SDS-PAGE 40 kda Western blot α 16 38% 18 93% 64 69% 24 5-6 0 Western Blot ELISA I 1-2 1 85% 1 1 3 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 2014-08-20 31328014 31400786 2012gjhz0009 GJHZ20130408174112021 JCYJ20130329110735981 JCYJ20120613173233810 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 LB DNA Eco R I Xho I ABCpred Prediction MEGA5 1 Clustalx1 83 1 2 2 Eco R I Xho I 2 pet-28a pmd18-t- 2 1 pet-28a pet-28a- 1 065 bp E coli Top10 354 NCBI http / /www ncbi nlm nih gov /gorf /gorf html 1 2 3 1 pet-28a- 37 200 r min - 1 1 1 000 IPTG 1 0 mol L - 1 125 r min - 1 8 h 1 ml 4 1 PBS 100 μl 20 μl 5 loading buffer 10 min 10 μl 10% SDS-PAGE pet Ni + 1 Ni + 2 2 pet-28a + - 1 2 4 1 Western pet-28a Eco R I Blot SDS-PAGE Xho I SDS-PAGE PVDF cdna 1 065 bp 5% TBS 37 2 h 2 5% TBST 4 2 20 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 average of hydropathicity - 0 322 TMHMM 2 4 1 Western Blot SDS-PAGE PVDF 20 Western Blot ECL 4 40 ku HNN α 16 38% β 18 93% 64 69% 6 2 5 2 AB- 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 OD 2 5 3 6 NCBI IgE Blastp Blomia tropicalis Ixodes scapularis Meta- seiulus occidentalis Saccoglossus kowa- 5 ELISA 2 5 268 ~ 283 279 ~ 291 294 ~ 309 305 ~ 320
104 2015 levskii Pediculus humanus corporis MEGA5 1 Clustalx1 83 Tribolium castaneum Crassostrea 7 gigas Danaus plexippus AAQ24554 1 XP - Dendroctonus ponderosae Daphnia pulex 002414267 1 Sus scrofa Fasta 6 7 3 11-13 Western Blot 11 ~ 100 kda 7 ELISA Westernblot Der f2 IgE 24 8 Der f1 α- 6 12 69 75 40 kd 9-10
1 105 4 8Eric 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 2003 112 1 79-86 J 9Seung Youp Shin Gyu Young Hur Young Min Ye et al A 2011 35 5532-535 2Masashi 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 2006 339 2 679-686 3Wayne R Thomas Belinda J HalesWendy-Anne Smith House dust mite allergens in asthma and allergy J Trends in Molecular Medicine 2010 16 7 321-328 4Susanne Vrtala HansHuberWayne R Thomas Recombinant house dust mite allergens J Methods 2014 66 1 67-74 634 5 Der p2 J 2009 25 8764-767 6 Der f15 J case of occupational rhinitis caused by porcine pancreatic extract developing into occupational asthma J J Korean Med Sci 2008 23 2 347-349 10Brant Hole Cannon et al Occupational asthma caused by cellulase and lipase in the detergent industry J Occup Environ Med 2004 61 793-795 11Yu 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 2014 7 3 630-12Kyoung 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 2014 6 3 196-200 13Bozek A Ignasiak B Filipowska B et al House dust mite 2013 37 2159-161 sublingual immunotherapy a double-blindplacebo-con- 7 16 trolled study in elderly patients with allergic rhinitis J J Clinical & Experimental Allergy 2012 43 2 242-248 2013 53 127-10 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 518060 China 2 Immunology Department of Nanchang University Medical School Nanchang Jiangxi 330006 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