RS21-C6分子的原核表达、纯化、抗体制备及免疫组织化学分析

目的:在原核细胞中表达胸腺发育相关分子RS21-C6重组蛋白,制备RS21-C6的多克隆抗体,研究RS21-C6分子在胸腺组织中的分布.方法: 在大肠杆菌中表达和纯化RS21-C6重组蛋白,免疫家兔制备多克隆抗体,以免疫组织化学实验分析RS21-C6分子在小鼠胸腺的分布.结果: 成功表达和纯化了RS21-C6重组蛋白,并制备获得高效价的特异性多克隆抗体.免疫组织化学显示RS21-C6分子在胸腺髓质区有较强的表达,在皮髓质交界处和皮质区也有散在分布.从胸腺细胞分布类型来看,RS21-C6分子共表达于胸腺淋巴细胞和基质细胞.结论: RS21-C6分子在胸腺分布广泛,且具有较高的表达水平,推测其在胸腺细胞发育的不同阶段均可发挥作用,是一个可能具有多种功能的胸腺发育相关分子.

Prokaryotic expression, purification and preparation of polyclonal antibody and immunohistochemistry analysis of RS21-C6 molecule

Objective: To express a thymocyte development related molecule RS21-C6 protein,prepare its polyclonal antibodies and study its distribution in thymus by immunohistochemistry analysis. Methods: RS21-C6 recombinant protein was expressed in E.coli system,and then purified by Ni-NTA chromatographic method . Polyclonal antibodies were prepared by immunized rabbits with the purified recombinant protein. Immunohistochemistry analysis was performed to show the distribution of RS21-C6 in mouse thymus tissue. Results: RS21-C6 recombinant protein was successfully expressed and purified. Then,the high titer polyclonal antibody was prepared and its specificity was confirmed. The immunohistochemistry analysis demonstrated that RS21-C6 had an intense expression in the medullary region of thymus,while scattered in the cortical region and the cortical-medullary junction region of thymus. It also showed that RS21-C6 was co-expressed in thymocyte and thymic stromal cells. Conclusion: RS21-C6 molecule is broadly and highly expressed in thymus,which suggests that this molecule may perform various functions in thymocytes at different developmental stages.