摘要
目的克隆表达及纯化屋尘螨第九组变应原(Der p9)蛋白,并鉴定其反应原性。方法提取屋尘螨总RNA,通过RT-PCR克隆Der P9基因,并连接到pET-32a表达载体上,得到的重组质粒pET-32a-Der p9转化至E.Coli BL21(DE3),用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过镍离子亲和层析纯化目的蛋白;通过Western blotting等方法检测Der p9重组蛋白的反应原性。结果 Der p9基因片段大小约为850 bp,其基因片段与Gen Bank公布的Der p9基因(登录号为AAP57077.1)同源性为81.94%;重组Der p9在BL21(DE3)中可高效表达,分子质量约为47 k Da,表达后的重组蛋白主要以包涵体的形式存在。Der p9重组蛋白与屋尘螨过敏患者血清呈阳性反应,具有反应原性。结论成功克隆并表达出Der p9,通过纯化获得较强反应原性的Der p9重组蛋白,为尘螨过敏性疾病诊断及免疫治疗奠定理论基础。
Objective To clone,express and purify the Dermatophagoides pteronyssinus allergen Der p9,and to identify its immunogenicity. Methods The total RNA was extracted from Dermatophagoides pteronyssinus.Der p9 gene was amplified by RT-PCR and linked to the pET-32a vector.The recombinant plasmids were transformed into E.coli BL21 (DE3) and induced byisopropyl-β-D-thiogalactopyranoside (IPTG) for protein expression.The recombinant protein was purified by Ni^+ chelating affinity chromatography and its immunogenicity was detected by Western blotting. Results The Der p9 gene fragment was approximately 850 bp in size and 81.94% homologous to GenBank AAP57077.1.The recombinant Der p9 protein was highly expressed in BL21 (DE3) in an inclusion body form with a molecular weight of 47 kDa.Western blot showed that the recombinant protein could react with the serum of dust mite-allergic patients. Conclusion The successful cloning,expression and purification of Der p9 with high immunogenicitylay the theoretical foundation for diagnosis and immunotherapy of dust mite-allergic diseases.
作者
李平
黄娜娜
常可欣
李诞
杨礼腾
刘志刚
刘晓宇
LI Ping;HUANG Na-na;CHANG Ke xin;LI Dan;YANG Li-teng;LIU Zhi-gang;LIU Xiao-yu(Institute of Allergy and Immunology,Shenzhen University,Shenzhen 518060,China;Department of Respiratory and Allergy,the Third Affiliated Hospital of Shenzhen University,Shenzhen 518001,China)
出处
《南昌大学学报（医学版）》
CAS
2018年第6期1-4,10,共5页
Journal of Nanchang University：Medical Sciences
基金
国家自然科学基金(31729002
81628001)
广东省科技计划项目(2014B090901041
2016A020216029)
深圳市孔雀团队项目(KQTD20170331145453160)
呼吸疾病国家重点实验室开放基金(SKLRD2018ZJ001)
广州市科学研究计划重点项目(201804020043)
深圳市科技计划基础研究项目(JCYJ20160429171931438
JCYJ20160429114659119)
关键词
屋尘螨
Derp9
表达
纯化
反应原性
Dermatophagoides pteronyssinus
Der p9
expression
purification
immunogenicity
