屋尘螨第21类过敏原原核表达和过敏原性鉴定
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摘要
目的屋尘螨(Dermatophagoides pteronyssinus)是引起过敏性疾病的主要过敏原,鉴定其免疫原性是研究哮喘和其他过敏性疾病的基础。由于不同地区生物的多样性,本实验在国内首次对屋尘螨Der p 21进行克隆、表达和免疫原性鉴定。方法提取屋尘螨总RNA,RT-PCR扩增Der p 21的cDNA片段,根据已知Der p 21序列设计出表达引物,再通过已获得的cDNA和引物进行PCR扩增,将扩增产物连接到克隆载体上并转入克隆菌E.coli Top10中,涂板过夜培养,挑选菌落,保菌,取样送至公司测序。将测序正确的基因转入表达载体PET32中,经酶切鉴定测序再转入表达菌BL21中,大量表达重组蛋白Der p 21经过纯化后,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测目的蛋白表达并进行免疫印迹分析(Western blotting)。结果双酶切显示Der p 21已成功连接在载体上,SDS-PAGE显示目标基因在大肠埃希菌BL21成功表达,纯化后的重组Der p 21蛋白分子量约为50 kD,Western blotting结果显示有明显条带。结论成功克隆表达Der p 21 cDNA,表达和纯化出重组蛋白,并具有免疫原性。
Objective To express the allergen(Der P 21) protein of house dust mite(HDM) 21 and to identify itsallergenicity. Methods The total RNA of HDMs was extracted, and the c DNA fragment of Der p 21 was amplified by RT-PCR. The expression primers were designed according to the known Der p 21 sequences, and then amplified by PCRamplification of the obtained cDNA and primers. The vector was cloned and transferred into the cloning E. coli TOP10, and theplate was cultured overnight, and the colonies were selected, preserved, and sampled and sent to the company for sequencing.The correctly sequenced gene was transferred into the expression vector pET-32, digested by restriction enzyme digestion andthen transferred into the expression strain BL21. After the expression and purification of the recombinant protein Der p 21, theexpression of the target protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) andimmunoblot analysis(Western blotting) was performed. Results Double enzyme digestion experiment showed that Der p 21 was successfully ligated to the vector. The results of SDS-PAGE showed that Der p 21 was successfully expressed in Escherichia coli TOP10. The molecular weight of recombinant Der p 21 was about 50 kD. Western blotting showed obviousbands. Conclusion Der p 21 cDNA was successfully cloned, and the recombinant protein was expressed and purified andshows allergenicity.
Objective To express the allergen(Der P 21) protein of house dust mite(HDM) 21 and to identify itsallergenicity. Methods The total RNA of HDMs was extracted, and the c DNA fragment of Der p 21 was amplified by RT-PCR. The expression primers were designed according to the known Der p 21 sequences, and then amplified by PCRamplification of the obtained cDNA and primers. The vector was cloned and transferred into the cloning E. coli TOP10, and theplate was cultured overnight, and the colonies were selected, preserved, and sampled and sent to the company for sequencing.The correctly sequenced gene was transferred into the expression vector pET-32, digested by restriction enzyme digestion andthen transferred into the expression strain BL21. After the expression and purification of the recombinant protein Der p 21, theexpression of the target protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) andimmunoblot analysis(Western blotting) was performed. Results Double enzyme digestion experiment showed that Der p 21 was successfully ligated to the vector. The results of SDS-PAGE showed that Der p 21 was successfully expressed in Escherichia coli TOP10. The molecular weight of recombinant Der p 21 was about 50 kD. Western blotting showed obviousbands. Conclusion Der p 21 cDNA was successfully cloned, and the recombinant protein was expressed and purified andshows allergenicity.
引文
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[13]HUI Y,LI L,QIAN J,et al.Efficacy analysis of three-year subcutaneous SQ-standardized specific immunotherapy in house dust miteallergic children with asthma[J].Experimental and Therapeutic Medicine,2014,7(3):630-634.
[14]JEONG K Y,LEE J H,KIM E J,et al.Current status of standardization of inhalant allergen extracts in Korea[J].Allergy Asthma Immunol Res,2014,6(3):196-200.
[15]PERZANOWSKI M S,PLATTS-MILLS T A.Further confirmation of the relevance of cockroach and dust mite sensitization to innercity asthma morbidity[J].Clin Exp Allergy,2009,39(9):1291-1293.
[16]LI J,WANG H Y,CHEN Y,et al.House dust mite sensitization is the main risk factor for the increase in prevalence of wheeze in 13-14 year old schoolchildren in Guangzhou city,China[J].Clinical&Experimental Allergy,2013,43(10):1171-11792018-10-08
[2]MASOLI M,FABIAN D,HOLT S,et al.The global burden of asthma:executive summary of the GINA Dissemination Committee report[J].Allergy,2004,59(5):469-478.
[3]CALDERON M A,DEMOLY P,GERTH VAN WIJK R,et al.EAA-CI:A European Declaration on Immunotherapy.Designing the future of allergen specific immunotherapy[J].Clin Transl Allergy,2012,2(1):20.
[4]LI J,SUN B,HUANG Y,et al.A multicentre study assessing the prevalence of sensitizations in patients with asthma and/or rhinitis in China[J].Allergy,2009,64(7):1083-1092.
[5]刘晓宇,吴捷,王斌,等.中国不同地理区域室内尘螨的调查研究[J].中国人兽共患病学报,2010,26(4):310-314.
[6]THOMAS W R,HALES B J,SMITH W A.House dust mite allergens in asthma and allergy[J].Trends Mol Med,2010,16(7):321-328.
[7]WEGHOFER M,DALL'ANTONIA Y,GROTE M,et al.Characterization of Der P 21,a new important allergen derived from the gut of house dust mites[J].Allergy,2008,63(6):758-767.
[8]PULSAWAT P,THEERAAPISAKKUN M,NONY E,et al.Characterization of the house dust mite allergen Der P 21 produced in Pichia pastoris[J].Protein Expression and Purification,2014,101:8-13.
[9]BILL R M.Recombinant protein subunit vaccine synthesis in microbes:a role for yeast?[J].Journal of Pharmacy and Pharmacology,2015,67(3):319-328.
[10]CHAN S L,ONG S T,ONG S Y,et al.Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group13 allergen as a hypoallergen[J].J Immunol,2006,176(8):4852-4860.
[11]THOMAS W R.The advent of recombinant allergens and allergen cloning[J].J Allergy Clin Immunol,2011,127(4):855-859.
[12]VRTALA S,HUBER H,THOMAS W R.Recombinant house dust mite allergens[J].Methods,2014,66(1):67-74.
[13]HUI Y,LI L,QIAN J,et al.Efficacy analysis of three-year subcutaneous SQ-standardized specific immunotherapy in house dust miteallergic children with asthma[J].Experimental and Therapeutic Medicine,2014,7(3):630-634.
[14]JEONG K Y,LEE J H,KIM E J,et al.Current status of standardization of inhalant allergen extracts in Korea[J].Allergy Asthma Immunol Res,2014,6(3):196-200.
[15]PERZANOWSKI M S,PLATTS-MILLS T A.Further confirmation of the relevance of cockroach and dust mite sensitization to innercity asthma morbidity[J].Clin Exp Allergy,2009,39(9):1291-1293.
[16]LI J,WANG H Y,CHEN Y,et al.House dust mite sensitization is the main risk factor for the increase in prevalence of wheeze in 13-14 year old schoolchildren in Guangzhou city,China[J].Clinical&Experimental Allergy,2013,43(10):1171-11792018-10-08