小鼠白细胞介素21原核表达载体的构建及活性鉴定

Construction of prokaryotic vector expressing murine interleukin-21 and identification of its activities

目的:白细胞介素21是近年发现的由T细胞分泌的细胞因子,具有广泛的免疫调节作用和抗肿瘤活性。为进一步观察其体内外免疫作用,拟构建小鼠白细胞介素21(mIL-21)原核表达载体,并对其表达的活性进行鉴定。方法:实验于2006-03/2007-05在南京医科大学附属南京儿童医院儿科研究所和东南大学基础医学院病原生物学与免疫学系完成。实验材料:实验中所用BALB/c小鼠由扬州大学比较医学中心提供,六七周龄,雌雄兼备,实验过程中对动物处置符合动物伦理学标准。pcDNA3.1/mIL-21质粒由东南大学基础医学院免疫学实验室提供、实验方法:以pcDNA3.1/mIL-21质粒为模板,PCR获得长度为369 bp的mIL-21基因片段。将其定向克隆至pET-28a(+)质粒中,采用PCR酶切和测序等方法对重组质粒进行鉴定。并对重组蛋白进行IPTG诱导表达、Western blot及体外生物学活性检测。结果:构建了重组原核表达载体pET-28a(+)/mIL-21,经鉴定后证实重组质粒构建正确。SDS-PAGE显示含重组质粒的诱导菌在M16 000位置出现一蛋白条带。Western blot结果显示该蛋白具有良好的免疫原性。体外生物学功能检测显示该重组蛋白能够增强小鼠脾细胞中的NK细胞杀伤活性。结论:成功构建了重组原核表达载体pET-28a(+)/mIL-21,表达的mIL-21具有良好的免疫原性。

AIM：lnterleukin-21 is a recently characterized T cell-derived cytokine with a broad immunoregulatory activity and can stimulate durable antitumour responses. This study aimed to construct a prokaryotic vector expressing murine interleukin-21 and identify its biological activity in order to provide with condition for further research of its immune function in vivo and vitro. METHODS： From March 2006 to May 2007, experiments were performed at Paediatric Research Institute, Nanjing Children＇s Hospital, Nanjing Medical University, and Department of Pathogenic Biology and Immunology, Southeast University. BALB/c mice of 6-7-week of age were obtained from Comparative Medicine Center of Yangzhou University, of either sex. The treatments on the animals accorded with the criteria of animal ethics. The pcDNA3.1/mlL-21 plasmid was provided by Laboratory of Immunology, School of Basic Medical Science, Southeast University. The gene of interleukin-21 was amplified from the recombinant plasmid pcDNA3.1/mlL-21 by polymerase chain reaction and cloned directly into pET-28a （＋） vector and form the prokaryotic expression vector pET-28a （＋）/mlL-21. It was identified by polymerase chain reaction, restriction enzyme digesting and DNA sequencing, respectively. The IPTG induced mlL-21 was detected by SDS-PAGE assay and the immune activity of mlL-21 was identified by Western-blot assay and the effect of mlL-21on NK activity in vitro was detected by flow cytometry. RESULTS： The results of restriction enzyme digesting and DNA sequencing assays showed the constructed vector was correct and was the same as the predicted result. SDS-PAGE result showed there was one more additional band in recombinant bacteria group than that of the control, which was about M16 000, just as predicted size. Western-blot assay showed that the expressed mlL-21 had binding activity with specific antibody. And the cytotoxicity of NK cells showed that it was enhanced by the expressed mlL-21. CONCLUSION： The prokaryotic expression vector pET-28a （＋）/mlL-21 is successfully constructed and the expressed mlL-21 has immunogenicity.