变应性鼻炎IgE高亲和力可溶型Fc段受体1α(sFcε1α)蛋白的制备及血清中相应抗体的检测  

Preparation of human soluble FcεR1α and detection of serum FcεR1α antibodies in patients with allergic rhinitis

作　　者：邢慧慧[1,2] 邵辉[3] 曹秀琴[4] 杨志伟[2] 

机构地区：[1]宁夏医科大学临床医学院检验系,宁夏银川750004 [2]宁夏医科大学基础医学院病原生物学与免疫学系,宁夏银川750004 [3]宁夏医科大学总医院银川医院,银川市第三人民医院,宁夏银川750004 [4]生育力保持省部级共建教育部重点实验室,宁夏银川750004

出　　处：《细胞与分子免疫学杂志》2016年第5期650-654,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81160375);中央地方共建创新团队开放课题(2015年);宁夏自然科学基金(NZ15232)

摘　　要：目的构建人可溶型Fc段受体1α(s FcεR1α)的原核表达载体,诱导表达并纯化重组FcεR1α胞外区段蛋白,检测其与血清中Ig E的结合力及相应抗体的含量。方法应用巢式PCR技术获得人FcεR1α胞外区段基因,构建原核表达载体p ETs Fcε1α,最佳诱导条件表达出s FcεR1α,采用亚胺二乙酸His标签纯化树脂纯化并进行Western blot法鉴定。ELISA检测人s FcεR1α与血清中Ig E的结合力及血清中s FcεR1α总量、s FcεR1α-Ig E及抗FcεR1α自身抗体的含量。结果扩增出人FcεR1α胞外区段基因,大小为600 bp左右。p ET-s FcεR1α经PCR、双酶切、测序鉴定正确。诱导表达、纯化出人s FcεR1α,相对分子质量(Mr)大小约为42 000。Western blot法鉴定为人s FcεR1α。人s FcεR1α可以与人血清中Ig E结合。变应性鼻炎(AR)患者血清中s FcεR1α总量、s FcεR1α-Ig E含量均低于正常人,抗FcεR1α抗体含量高于正常人。结论获得人s FcεR1α,s FcεR1α与人血清中Ig E有较强的结合力,AR患者血清中s FcεR1α总量、s FcεR1α-Ig E含量均低于正常人,抗FcεR1α抗体含量高于正常人。Objective To induce the expression of human soluble Fc epsilon receptor I alpha （FcεR1α） in a prokaryotic expression vector, purify the recombinant human FcεR1α protein, detect its binding affinity for human serum IgE antibodies and detect the levels ofFcεR1α, sFcεR1α-IgE and FcεR1α antibodies. Methods The FcεR1α extracellular region gene was amplified using nested polymerase chain reaction （PCR） and was expressed in a prokaryotic expression vector pET-sFcεR1α using recombinant DNA technology under optimal conditions. The human sFcεR1α protein was purified using iminodiacetic acid （IDA） His binding resin and identified using Western blotting. The affinity between the recombinant human sFcεR1α and serum IgE antibodies and the levels of total sFcεR1α, sFcεR1α-IgE and FcεR1α antibodies were measured using ELISA. Results The amplified gene corresponding to the extracellular region FcεR1α was approximately 600 bp. PCR, double enzyme digestion and sequencing confirmed the correct sequence of the expression vector pET-sFcεR1α. After human sFcεR1α protein was induced in the expression vector pET-FcεR1α and purified, Westem blotting showed that its relative molecular mass （Mr） was approximately 42 000. ELISA revealed that the human sFcεR1α bound with a high affinity to serum IgE, and the lower levels of total sFcεR1α and sFcεR1α-IgE and higher levels of serum anti-FcεR1α antibodies in the patients with allergic rhinitis than in the normal subjects. Conclusion We successfully synthesized human sFcεR1α which had a strong binding affinity for human serum IgE. The higher levels of serum anti-FcεR1α antibodies in the patients with allergic rhinitis than the normal subjects.

关 键 词：IG E高亲和力Fc段受体1α(FcεR1α) 可溶型 结合力 自身抗体 

分 类 号：R392.11[医药卫生—免疫学] R765.21[医药卫生—基础医学]