论文部分内容阅读
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 construct prokaryotic expression vector of human soluble Fc fragment receptor 1α (s FcεR1α), and to induce the expression and purification of the recombinant extracellular domain of FcεR1α. The binding capacity of IgF and its corresponding antibody in serum was detected. Methods The extracellular domain of human FcεR1α gene was obtained by nested PCR. The prokaryotic expression vector p ETs Fcε1α was constructed. The sIFεR1α was expressed under the best induction conditions. The recombinant plasmid was purified by His-tagged imine diacetate and identified by Western blot. ELISA was used to detect the binding of human s FcsR1α to serum IgE and the total amount of sFεR1α, sFcR1α-IgE and anti-FcsR1α autoantibodies in serum. Results The human extracellular domain of human FcεR1α gene was amplified with a size of 600 bp. p ET-s FcεR1α by PCR, double enzyme digestion, sequencing identification correct. Induced expression, purified human FcεR1α, the relative molecular mass (Mr) size of about 42 000. Western blot was identified as human s FcεR1α. Human s FcεR1α binds to Ig E in human serum. The total amount of sFεR1α and sFcεR1α-IgE in serum of patients with allergic rhinitis (AR) were lower than that of normal people, and the content of anti-FcsR1α antibody was higher than that of normal people. CONCLUSIONS: Human sFcR1α, s FcεR1α have strong binding affinity with human IgE. The total amount of sFεR1α and sFεR1α-IgE in serum of AR patients is lower than that of normal people, and the content of anti-FcsR1α antibody is higher than that of normal people .