小鼠透明质酸受体RHAMM的载体构建、诱发表达和产物特性分析

Construction or Wild and Mutant Murine RHAMM Harboring Vectors: Induction of Gene Expression and Characterization of Purified Recombinant Proteins

目的RHAMM作为透明质酸受体参与肿瘤细胞的恶性转化、侵袭和转移。根据RHAMMcDNA序列在人、大鼠和小鼠间有高度保守性的遗传学特点，构建诱发表达质粒，以期制备人／鼠共识别的RHAMM生物和免疫制剂。方法利用己克隆的野生型和突变型RHAMM片段构建了可被IPTG诱发表达的pGEX-4TRHAMMw和RHAMMm质拉.并用两者转化大肠杆菌。结果在筛选和大规模培养转化细胞后，访导并纯化出分子量分别为37kD和53kD的RHAMMw和RHAMMm-GST融合蛋白。使用兔抗鼠RHAMMw／m抗体所做的Western印记杂文显示，该抗体既可识别这两种蛋白，也能检测到人胃癌和正常胃粘膜上皮间RHAMM基因的差异表达。结论本研究构建的两种诱发表达质粒，为RHAMM与人类疾病关系的研究提供了分子和免疫制剂。

Objective RHAMM is a newly cloned hyaluronan receptor which has been known to be important in malingnant transformation,invasion and metastasis. It is aimed in this study to prepare the molecular reagents for HRAMM study. Methods The cloned wild and mutant RHAMM cDNA fragments were constructed into GST combining region of pGEX-4T-2 vector. The recombinant protein expression was inducted by treating the transformed cells with 0.1mmol/L lPTG. After protein purification,the antigenic properties of wild and mutant RHAMM fusion proteins were checked by Western blot analyses, using a rabbit anti-mouse antibody against the conserved domains of the targct proteins. Results The pGEX-4T-2 vectors harboring wild or mutant RHAMM sequence were constructed successfully, which were named as pGEX-4T/RHAMMw and pGEX-4T/RHAMMm. Sufficient amounts of RHAMMw and RHAMMm fusion proteins in the molecular weights of 37kD and 53kD were prepared.which can be recognized by the rabbit anti-mouse RHAMMw/m polyclonal antibody. Conclusion The molecular reagents obtained from this study would have potential value in highlighting the role (s ) of RHAMM in human diseases.