1.Health Science Center, Shenzhen University, Shenzhen 518060, China; 2.Department of Allergy, the Third Affiliated Hospital of Shenzhen University, Shenzhen 518060, China
Abstract:Blattellagermanica contains many allergens in its secretions, excretions, molting products and corpses, among which Blag 5 is an important allergen causing allergic diseases. Object:Here, we used E.coli to clone and express Blattellagermanica allergen Blag 5, and then used serum IgE from patients with allergic diseases to test its allergenicity. Bioinformatics was used to predict the role of the Blag 5 gene and understand its molecular characteristics. Method: First, the total RNA of Blattellagermanica was extracted, and gene-specific primers were designed according to the known Blag 5 gene sequence (GenBank: U92412). The cDNA was amplified and ligated with a restriction endonuclease to construct the expression vector pET-28a(+)-Bla g 5, which was then transformed into Rosetta (DE3) competent cells. Monoclonal positive colonies were selected for amplification and culture. The expression of the target protein was induced with 0.1 mm/mol IPTG, the expressed product was purified with metal chelate chromatography, and the immunological properties of the purified product were identified by western blotting. Result:The expressed product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of soluble expressed Bla g 5 protein was approximately 25 kDa, in agreement with the theoretical results. The immunoblotting results of the purified recombinant protein showed that Bla g 5 specifically binds serum IgE in allergic patients. On the basis of the gene sequence, we predicted the secondary and three-dimensional structure, hydrophilicity, plasticity, antigenicity, surface accessibility and homology. The results indicated characteristics typical of glutathione S-transferase. Conclusion:The heterologous expression of German cockroach Blag5 allergen protein in this study indicated high binding activity to the serum of allergic patients, thus laying a foundation for the development of diagnostic reagents and vaccines for allergic diseases.
[1] Schapheer C, Sandoval G, Villagra CA.Pest cockroaches May overcome environmental restriction due to anthropization[J]. J Med Entomol,2018,55(5):1357-1364. DOI: 10.1093/jme/tjy090 [2] Orlandi RR, Kingdom TT, Hwang PH.International consensus statement on allergy and rhinology: rhinosinusitis executive summary[J]. Int Forum Allergy Rhinol. 2016,6(S 1): 3-21. DOI: 10.1002/alr.21694 [3] Chan TF, Ji KM, Yim AK, et al.The draft genome, transcriptome, and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens[J]. J Allergy Clin Immunol,2015,135(2):539-548.DOI:10.1016/j.jaci. 2014.09.031 [4] 季文霞,谯承德,陈泽金,等.四川绵阳地区变应原皮肤点刺试验308例临床分析[J].中国耳鼻咽喉头颈外科,2013,20(8):423-425. DOI:10.16066/j.1672-7002.2013.08.017 [5] 李春林,谢伟伟,刘硕,等.海南省500例蟑螂过敏皮肤点刺试验分析[J].海南医学,2011,22(10):16-17. DOI:10.3969/j.issn.1003-6350.2011.10.006 [6] Lee CY, Hemingway J, Yap HH, et al.Biochemical characterization of insecticide resistance in the German cockroach, Blattella germanica, from Malaysia[J]. Med Vet Entomol,2000,14(1):11-8. DOI: 10.1046/j.1365-2915.2000.00215.x [7] Wu X, Appel AG.Insecticide Resistance of Several Field-Collected German Cockroach (Dictyoptera: Blattellidae) Strains[J]. J Econ Entomol,2017,110(3):1203-1209. DOI:10.1093/jee/tox072 [8] Lin JL, Wang YY, Xiao XJ, et al.Characterization of a new subtype of allergen in dermatophagoides farinae-Der f 28[J]. J Thorac Dis,2015,7(10):1842-1849. DOI:10.3978/j.issn.2072-1439.2015.10.43 [9] Arruda LK, Vailes LD, Platts-Mills TAE, et al.Induction of IgE antibody responses by glutathioneS-Transferase from the German Cockroach (Blattellagermanica)[J]. J Biolog Chem, 1997, 272(33):20907-20912. DOI: 10.1074/jbc.272.33.20907 [10] Jeong KJ, Jeong KY, Kim CR, et al.IgE-Binding Epitope Analysis of Bla g 5, the German Cockroach Allergen[J]. Protein Peptide Letters, 2010, 17(5):573-577. DOI : 10.2174/092986610791112765 [11] Lin YC, Su HJ, Hsiue TR, et al.Levels of house dust mite-specific IgE and cockroach-specific IgE and their association with lower pulmonary function in Taiwanese children[J].Chest,2002,121:347-353. DOI: 10.1378/chest.121.2.347 [12] Bozza S, Gaziano R, Lipford GB, et al.Vaccination of mice against invasive aspergillosis with recombinant Aspergillus proteins and CpG oligodeoxynucleotides as adjuvants[J]. Microbes Infect, 2002, 4(13):1281-1290. DOI: 10.1016/s1286-4579(02)00007-2 [13] 常维佳,姜玉新,陈文魁,等.重组粉尘螨Ⅱ类变应原的原核表达及纯化[J].中国病原生物学杂志, 2012,7(2):121-123. DOI:10.13350/j.cjpb.2012.02.012 [14] Huang J, Honda W.CED: a conformational epitope database[J]. BMC Immunology, 2006, 7(1):7-10. DOI: 10.1186/1471-2172-7-7