Liu Hong-chen, Mao Chao-ming, Su Xiao-yu, Ruan Zheng, Ding Qiu-lan, Wang Xue-feng, Wang Hong-li, Xi Xiao-dong
State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Jul;25(7):619-22.
To prepare anti-c-Kit monoclonal antibodies and characterize their specificity of epitope recognition.
cDNA encoding human c-Kit extracellular domain was constructed into a procaryotic expression vector pQE30 and the correctness of the reconstructed plasmid pQE30-KitD4-5 was verified by sequencing. The plasmid was transformed into E.coli M15 strain. Recombinant 6 x His pQE30-KitD4-5 was expressed after induction by IPTG for 4 h. Then SDS-PAGE results suggested that the products mainly formed inclusion bodies. The fusion protein was further purified with Ni-NTA-His affinity chromatography and then used to immunize BALB/c mice. The hybridomas were achieved by fusing the immunized spleen cells with the Sp2/0 myeloma cell line. The positive clones were screened by FCM with CHO-hKit cells. Hybidoma clones secreting anti-c-Kit antibodies were further subcloned and investigated for their biological activities by Western blot, rapid isotyping analysis and FCM.
Recombinant human c-Kit fusion proteins were in vitro expressed and purified to be used as immunogen. One stable hybridoma cell line, which continuously secrets specific anti-c-Kit monoclonal antibody ((SRJ1)) was established. The biological activity studies showed that the monoclonal antibody recognized the natural c-Kit expressed on the Kasumi leukemia cell line, but failed to bind to the normal human peripheral blood cells. Interestingly, this monoclonal antibody failed to recognize a subpopulation of Kasumi cells that is reactive with the commercial anti-c-Kit mAb Ab81 suggesting that the c-Kit expressed by this subpopulation contains some sequencial and/or structural aberrations that are distinguishable by mAb SRJ1.
With an immunization procedure using purified recombinant human c-Kit fusion proteins. a hybridoma cell line continuously and stably secreting anti-c-Kit monoclonal antibody has been established. The monoclonal antibody SRJ1 specifically recognizes human c-Kit expressed on the leukemia cells, and may provide a novel approach to analyze the possible structural variations of c-Kit expressed by different cells.
制备抗c-Kit单克隆抗体并鉴定其表位识别特异性。
将编码人c-Kit胞外区的cDNA构建到原核表达载体pQE30中，通过测序验证重组质粒pQE30-KitD4-5的正确性。将该质粒转化到大肠杆菌M15菌株中。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导4小时后表达重组6×His pQE30-KitD4-5。随后十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果表明产物主要形成包涵体。用镍-亚氨基二乙酸-组氨酸（Ni-NTA-His）亲和层析进一步纯化融合蛋白，然后用于免疫BALB/c小鼠。通过将免疫的脾细胞与Sp2/0骨髓瘤细胞系融合获得杂交瘤。用表达人c-Kit的中国仓鼠卵巢细胞（CHO-hKit）通过流式细胞术（FCM）筛选阳性克隆。进一步亚克隆分泌抗c-Kit抗体的杂交瘤克隆，并通过蛋白质免疫印迹法（Western blot）、快速分型分析和流式细胞术研究其生物学活性。
体外表达并纯化重组人c-Kit融合蛋白用作免疫原。建立了一个稳定的杂交瘤细胞系，其持续分泌特异性抗c-Kit单克隆抗体（（SRJ1））。生物学活性研究表明，该单克隆抗体识别在Kasumi白血病细胞系上表达的天然c-Kit，但不与正常人外周血细胞结合。有趣的是，该单克隆抗体不能识别Kasumi细胞的一个亚群，该亚群与市售抗c-Kit单克隆抗体Ab81反应，这表明该亚群表达的c-Kit包含一些可被单克隆抗体SRJ1区分的序列和/或结构异常。
通过使用纯化的重组人c-Kit融合蛋白的免疫程序，建立了一个持续稳定分泌抗c-Kit单克隆抗体的杂交瘤细胞系。单克隆抗体SRJ1特异性识别白血病细胞上表达的人c-Kit，并可能为分析不同细胞表达的c-Kit的可能结构变异提供一种新方法。
