[摘要] 目的：制备鼠源粒细胞-巨噬细胞集落刺激因子(mouse granulocyte-macrophage colony stimulating factor，mGM-CSF)与促性腺激素释放激素(gonadotropin releasing hormone，GnRH)的融合蛋白mGM-CSF-GnRH3（mGGn）和mGM-CSF与胃泌素释放肽（gastrin-releasing peptide，GRP）的融合蛋白mGM-CSF-GRP6（mG6），探讨这两种融合蛋白在体外对黑色素瘤B16F10 细胞的抑制效果，并对其等电点、相对分子质量、疏水性、稳定性、亚细胞定位、信号肽、空间结构、潜在抗原表位等进行初步预测。方法：mGGn与mG6 融合蛋白制备成功后，通过显微观察、划痕实验、CCK-8 法、流式细胞术分别检测不同浓度蛋白对B16F10 细胞形态、细胞迁移、细胞增殖、细胞周期的影响，利用蛋白质在线分析系统EXPASY、GOR4、SWISS MODEL对重组融合蛋白进行基本属性、二三级结构分析预测，运用IEDB和ABCpred软件综合预测其Ｂ细胞抗原表位，采用SYFPEITHI、BlMAS和NetCTL软件综合预测其CTL表位，利用NetMHCIIpan 3.1 Server 和IEDB软件综合预测其Th 表位。结果：mGGn和mG6 融合蛋白均抑制肿瘤细胞的增殖和迁移；mGGn能使B16F10 细胞周期阻滞于G1 期，mG6 能使B16F10 细胞周期阻滞于S期，不能进入G2 期，从而抑制瘤细胞的增殖。mGGn和mG6 结构丰富，含有较多潜在的B细胞、CTL和Th表位。结论：mGGn与mG6 融合蛋白在体外对黑色素瘤B16F10细胞具有抑制作用，其生物信息学预测为进一步研究两种融合蛋白的生物学功能和免疫活性奠定了基础。

[Abstract] Objective: To prepare the fusion protein mGM-CSF-GnRH3 (mGGn) of mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) combining with gonadotropin releasing hormone (GnRH) and the fusion protein mGM-CSF-GRP6 (mG6) of mGM-CSF combining with gastrin-releasing peptide (GRP), and to investigate the inhibitory effect of the above two fusion proteins on B16F10 melanoma in vitro as well as to preliminarily predict their isoelectric point, relative molecular weight,hydrophobicity,stability,subcellular localization, signal peptide, spatial structure and potential epitopes. Methods: After the successful preparation of mGGn and mG6, the effects of different concentrations of fusion proteins on tumor cell morphology, migration, proliferation and cell cycle were detected by microscopic observation, scratch test, CCK-8 method and flow cytometry, respectively. The protein online analysis systems EXPASY, GOR4, SWISS MODEL were used to predict the basic properties and secondary/tertiary structure of recombinant fusion proteins.The B cell epitopes were predicted by IEDB and ABCpred software, the CTL epitopes were comprehensively predicted by SYFPEITHI,BlMAS and NetCTL software, and the Th epitopes were predicted by NetMHCIIpan 3.1 Server and IEDB software. Results:Both mGGn and mG6 inhibited the migration and proliferation of tumor cells. mGGn could block B16F10 cell cycle at G1 phase while mG6 could block B16F10 cell cycle at S phase, all of which prevented cells entering into G2 phase to inhibit tumor cell growth. The mGGn and mG6 fusion proteins got diverse structures and had multiple potential B epitopes, CTL epitopes and Th epitopes.Conclusion:mGGn and mG6 have inhibitory effect on B16F10 melanoma in vitro, and bioinformatics predictions have laid a foundation for further study of the biological functions and immunological activities of these fusion proteins.

国家自然科学基金资助项目（No.81172973，81373232）；国家高新技术研究发展（863）计划资助项目（No.2015AA020314）；国家级大学生创新创业训练计划资助项目（No.J1030830）；江苏高校优势学科建设工程资助项目（No.PAPD1）