霜天蛾昆虫变应原多克隆抗体的制备及免疫特性分析

Preparation and immune characteristic analysis of polyclonal antibody against Psilogramma menephron allergen

目的制备兔抗霜天蛾变应原多克隆抗体,分析其与特异性过敏患者血清抗体成分的异同,为免疫筛选cDNA文库,制备基因重组霜天蛾变应原奠定基础。方法应用霜天蛾变应原提取液免疫家兔获得霜天蛾变应原多克隆抗体,用酶联免疫吸附试验(ELISA)法测定混合免疫兔血清抗体效价;应用免疫印迹的方法对比分析免疫兔血清与特异性过敏患者血清抗体成分的异同。结果混合免疫兔血清效价经ELISA检测,效价>1:10000,免疫印迹检测兔抗血清免疫球蛋白G(IgG)可以特异性识别霜天蛾10种蛋白组分,相对分子质量分别为100000、92000、79000、74000、66000、49000、43000、36000、25000和16000;患者血清IgG可以识别6种蛋白组分,相对分子质量分别为79000、74000、66000、49000、36000和25000。结论制备出的兔抗血清与特异性霜天蛾过敏患者血清特异性识别霜天蛾变应原的结果接近,比患者可以识别更多的蛋白条带,可以应用免疫兔血清作为筛选霜天蛾cDNA文库的探针及重组霜天蛾变应原表达蛋白的鉴定分析。

Objective To prepare polyclonal antibody of the rabbit anti-Psilogramma menephron allergen and analyse the similarities and differences of the antibody components about rabbit serum and patients' serum with specific Psilogramma menephron allergy.The study provided the bases for immunoscreening Psilogramma menephron cDNA library and preparing the recombinant Psilogramma menephron allergen.Methods The polyclonal antibody was obtained by immunizing rabbits with Psilogramma menephron extracts.And its valence was tested with ELISA.The similarities and differences of the antibody components about rabbit serum and patients' serum with specific Psilogramma menephron allergy were tested with immunoblotting.Results The valence of mixed rabbits antiserum was more than 1∶10 000 with ELISA.The IgG-immunoblotting analysis of rabbit antiserum showed that the protein bands of molecular mass were 100 000,92 000,79 000,74 000,66 000,49 000,43 000,36 000,25 000,16 000;The results of analyzing 9 patients' sera with allergic to Psilogramma menephron showed that the protein bands of molecular mass were 79 000,74 000,66 000,49 000,36 000,25 000.Conclusions The prepared rabbit antiserum recognized the similar protein bands of Psilogramma menephron allergen with specific allergic patients' serum and it could combine more IgG bands than patients'.The immunized rabbit serum can be used as probe for immunoscreening Psilogramma menephron cDNA library and be used in identifying the expressed recombinant allergen of Psilogramma menephron.