mGM-CSF-GnRH3与mGM-CSF-GRP6融合蛋白体外抗肿瘤作用及生物信息学预测  

In vitro anti-tumor effect of mGM-CSF-GnRH3 and mGM-CSF-GRP6 recombinant fusion protein and their bioinformatics prediction

作　　者：刘淑君 韦晓芳 刘生凤 黄滢霜 张焱 曹荣月[1] LIU Shujun;WEI Xiaofang;LIU Shengfeng;HUANG Yingshuang;ZHANG Yan;CAO Rongyue(School of Life Science and Technol- ogy, China Pharmaceutical University, Nanjing 21009, Jiangsu, China)

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《中国肿瘤生物治疗杂志》2018年第6期582-589,共8页Chinese Journal of Cancer Biotherapy

基　　金：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)~~

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,m GM-CSF)与促性腺激素释放激素(gonadotropin releasing hormone,Gn RH)的融合蛋白m GM-CSF-Gn RH3(m GGn)和m GM-CSF与胃泌素释放肽(gastrin-releasing peptide,GRP)的融合蛋白m GM-CSF-GRP6(m G6),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:m GGn与m G6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、Bl MAS和Net CTL软件综合预测其CTL表位,利用Net MHCIIpan 3.1 Server和IEDB软件综合预测其Th表位。结果:m GGn和m G6融合蛋白均抑制肿瘤细胞的增殖和迁移;m GGn能使B16F10细胞周期阻滞于G1期,m G6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。m GGn和m G6结构丰富,含有较多潜在的B细胞、CTL和Th表位。结论:m GGn与m G6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。Objective： To prepare the fusion protein m GM-CSF-Gn RH3（m GGn） of mouse granulocyte-macrophage colony stimulating factor（m GM-CSF） combining with gonadotropin releasing hormone（Gn RH） and the fusion protein m GM-CSF-GRP6（m G6） of m GM-CSF combining with gastrin-releasing peptide（GRP）, and to investigate the inhibitory effect of the above two fusion proteins on B16 F10 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 m GGn and m G6, 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, Bl MAS and Net CTL software, and the Th epitopes were predicted by Net MHCIIpan 3.1 Server and IEDB software. Results：Both m GGn and m G6 inhibited the migration and proliferation of tumor cells. m GGn could block B16 F10 cell cycle at G1 phase while m G6 could block B16 F10 cell cycle at S phase, all of which prevented cells entering into G2 phase to inhibit tumor cell growth. The m GGn and m G6 fusion proteins got diverse structures and had multiple potential B epitopes, CTL epitopes and Th epitopes. Conclusion： m GGn and m G6 have inhibitory effect on B16 F10 melanoma in vitro, and bioinformatics predictions have laid a foundation for further study of the biological functions and immunological activities of these fusion proteins.

关 键 词：鼠源粒细胞-巨噬细胞集落刺激因子 促性腺激素释放激素 胃泌素释放肽 黑色素瘤 B16F10细胞 生物信息学分析 

分 类 号：R392.11[医药卫生—免疫学] R730.3[医药卫生—基础医学]