摘要
目的:利用基因工程技术制备具有双重生物学活性的MIP-2γ/GM—CSF融合蛋白。方法:通过引物设计和分级克隆的策略构建了MIP-2γ/GM—CSF融合基因真核表达载体,MIP-2γ与GM-CSF由(Gly4Ser)3相连;瞬时转染293-T细胞后收集含融合蛋白的细胞培养上清;研究了该融合蛋白在体外的促造血细胞增殖活性和对免疫细胞的趋化活性。结果:MIP-2γ/GM—CSF融合基因载体构建正确;融合蛋白分子量约为24.9 kD;初步生物学功能研究表明,融合蛋白能有效促进TF—1细胞增殖,比活性约为2.2×107IU/mg,并能显著刺激骨髓细胞的集落形成;融合蛋白能有效趋化未成熟树突状细胞和CD3+T细胞。结论:MIP-2γ/GM—CSF融合蛋白具有促进造血和趋化免疫细胞的双重活性,有望成为在造血调控和抗肿瘤免疫中具有良好开发应用前景的新型细胞因子。
Objective: To generate a new kind of cytokine MIP-2γ/GM-CSF fusion protein by genetic engineering technology and investigate its biological bifunction. Methods: The eukaryotic expressing vector for MIP-2γ/GM-CSF fusion protein was constructed by primer design and step by step doing. MIP-2-y gene and GM-CSF gene were linked via a linker sequence( Gly4Ser)3. After transient expression, the cell supernatants of 293-T cells containing the aimed protein were obtained. The hematopoietic and chemotactic activities of the fusion protein were investigated in vitro. Results: MIP-2γ/ GM-CSF fusion gene vector was confirmed by restriction analysis and sequencing analysis. The molecular weight of the protein was about 24. 9 kD. MIP-2γ/GM-CSF fusion protein could stimulate the proliferation of TF-1 cells like GM-CSF. The specific activity of the fusion protein was 2. 2 × 107IU/mg. The fusion protein was found to be effective in stimulating bone marrow cell colony-formation and chemoattracting immature dendritic cells and CD3+T cells. Conclusion: MIP-2γ/ GM-CSF fusion protein exhibits bifunctions which including the promotion of hematopoiesis and chemoattration of immune cells, suggesting that the fusion protein can be used to regulate hematopoiesis and antitumor immune response as a new kind of cytokine.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2004年第2期133-137,共5页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基因项目(30130170
39970839)
国家973免疫学项目(2001CB510002)
国家863项目(2001AA215091)
