论文部分内容阅读
【目的】囊素(BS)是禽类和哺乳动物中具有重要免疫调节功能的多肽,能有效促进杂交瘤细胞抗体的分泌,为探讨杂交瘤细胞是否有BS受体分子的表达。【方法和结果】本研究采用荧光显微镜、激光扫描共聚焦显微镜和流式细胞仪分析的方法,检测BS与杂交瘤细胞的结合特性。实验结果证实BS与杂交瘤细胞的结合具有特异性、趋饱和性和可逆性。为进一步分析BS与杂交瘤细胞的结合位点,实验中以BS分子作为靶标,对噬菌体随机12肽库进行了4轮亲和筛选,ELISA和竞争抑制试验显示2个噬菌体克隆能特异性与BS结合。对阳性噬菌体克隆进行序列测定分析表明,其插入的12肽分别为:ACTKHLCLLQPL、MSCNDTLCLLPN,保守序列为LCLL。体外实验表明,2个人工合成的12均都能在一定程度上抑制BS与杂交瘤细胞的特异性结合。【结论】本研究表明杂交瘤细胞具有BS结合的受体,这为进一步研究BS促杂交瘤细胞抗体分泌的信号传导通路奠定了基础。
【Objective】 Cystin (BS) is a polypeptide with important immunomodulatory function in poultry and mammals, which can effectively promote the secretion of antibody of hybridoma cells. To explore whether BS receptor molecule expression exists in hybridoma cells. Methods and Results In this study, the binding characteristics of BS and hybridoma cells were examined by fluorescence microscopy, laser scanning confocal microscopy and flow cytometry. The experimental results confirmed the binding of BS to hybridoma cells with specificity, tenacity and reversibility. In order to further analyze the binding sites of BS with hybridoma cells, 4 rounds of affinity screening of phage random 12-peptide library was performed using BS as a target. ELISA and competitive inhibition assays showed that the two phage clones were specific to BS Combined. Sequence analysis of the positive phage clones showed that the inserted 12-mer were: ACTKHLCLLQPL, MSCNDTLCLLPN and the conserved sequence was LCLL. In vitro experiments showed that both of the two synthetic 12 can inhibit the specific binding of BS to hybridoma cells to a certain extent. 【Conclusion】 This study shows that the hybridoma cells have BS-binding receptors, which lays the foundation for further study on the signal transduction pathway of BS-producing hybridoma cells.