FcεRⅠ受体α亚基的重组表达、单克隆抗体的制备及特异性鉴定

目的通过基因重组的方法表达IgE高亲和力受体FcεRⅠα亚基，并用重组蛋白制备单克隆抗体。方法用RT-PCR的方法从过敏性疾病病人外周血嗜碱性粒细胞调取FcεRⅠα蛋白基因，经T-A克隆、亚克隆至pET28a（＋）原核表达载体，将重组质粒转化到大肠杆菌BL．21．STAR（DE3），IPTG诱导表达FeaRIOt亚基蛋白，通过Ni-NTA亲和层析纯化融合蛋白；并用其免疫BALB／c小鼠，运用杂交瘤技术制备抗FeaRIOt亚基单克隆抗体，用细胞免疫荧光法对单克隆抗体的特异性进行鉴定。结果成功调取了人IgE高亲和力受体FcεRⅠα亚基的基因，且测序正确；构建原核表达质粒FcεRⅠα-pET28a（＋）；成功建立4株稳定分泌抗FcεRⅠα亚基的单克隆抗体杂交瘤细胞株，分别命名为G101、C22、G113、G42。通过细胞免疫荧光实验证实，4株单克隆抗体均能特异性结合人嗜碱性粒细胞表面的FcεRⅠα亚基。结论成功利用基因重组技术制备了FcεRⅠα亚基蛋白，制备了4株效价高、特异性好的抗FcεRⅠα亚基单克隆抗体，为FcεRⅠα亚基及其抗体在过敏性疾病中的生物学功能研究奠定了基础。

Prokaryotic Expression of FcεRⅠα Chain and Preparation and Characterization of the Monoclonal Antibody against FcεRⅠα Chain

Objective To prepare FcεRⅠα chain antigen with fusion protein of 6xHis by pET28a （＋） expression system, and the corresponding monoclonal antibodies （McAb）. Methods FcεRⅠα cDNA was obtained by RT-PCR from peripheral blood mononuclear cell of an allergic disease patient and cloned into the vector pET28a （＋）.The recombinant plasmid FcεRⅠα-pET28a （＋）was transferred into E.coli BL-21-STAR （DE3） and the 6xHis- FcεRⅠα fusion protein was expressed by inducing with IPTG. The fusion protein was purified by Ni Sepharose 6Fast Flow affinity chromatography. BALB/c mice were immunized with recombinant FcεRⅠα chain. Hybridoma cell lines were constructed from the immunized mice. The monoclonal antibodies were identified by cell immunofluoreseence. Resulits The cloned FcεRⅠα chain cDNA was confirmed by DNA sequencing. Fusion protein expressed as EB and accounted for about 15% of the total bacterial proteins. Four hybridoma cell lines that stably secreted McAbs against FcεRⅠα chain were obtained, which were named as G101, C22, G113 and G42. All the four McAbs could bound to FcεRⅠα chain obtained from basophil. Cell immunofluorescence showed that the antibody bound with human FcεRⅠα chain. Conclusion Monoclonal antibodies against FcεRⅠα chain with high titers and specificity have been successfully prepared, which has laid the foundation for further study ofFcεRⅠαchain.