人源R-spondin 2蛋白的生物信息学分析、表达、纯化及活性检测  

Bioinformatics analysis,expression,purification and activity detection of human R-spondin 2 protein

作　　者：耿腾洁 许剑锋[1,2] GENG Tengjie;XU Jianfeng(College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海农业部水产品贮藏保鲜质量安全风险评估实验室,上海201306

出　　处：《中国生物制品学杂志》2024年第9期1043-1049,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金(82173731)。

摘　　要：目的构建人源R-spondin 2(roof plate-specific spondin 2,RSPO2)的真核表达载体,表达、纯化融合蛋白RSPO2-Fu-Fc,用3C蛋白酶切除Fc结构域后,检测RSPO2-Fu的生物学活性。方法基于生物信息学方法分析人源RSPO2蛋白的理化性质和结构,筛选获得可在溶液中稳定存在的RSPO2截短蛋白RSPO2-Fu。将编码RSPO2-Fu蛋白的基因亚克隆至pCMV-Fc载体后,将质粒pCMV-RSPO2-Fu-Fc瞬时转染入HEK293F细胞中表达72 h,通过Protein A层析柱纯化融合蛋白RSPO2-Fu-Fc,用3C蛋白酶切去Fc结构域,分子筛进一步提纯获得RSPO2-Fu蛋白。最后,通过M50Super 8×TOPFlash检测RSPO2-Fu蛋白的生物学活性。结果生物信息学分析结果显示,RSPO2全长有243个氨基酸,相对分子质量为28300,等电点为9.42,为亲水性不稳定蛋白,RSPO2-Fu截短蛋白在溶液中的稳定性得到了极大提高;重组表达质粒pCMV-RSPO2-Fu-Fc经菌落PCR及测序证明构建正确;获得了纯度达95%的RSPO2-Fu蛋白;生物学活性检测结果显示,RSPO2-Fu的EC50值为1.5×10^(-12)mol/L。结论对RSPO2蛋白适当截短后,可获得稳定表达的RSPO2-Fu蛋白,其对Wnt信号通路有明显激活作用,为Wnt信号通路中RSPO2相关生物学研究提供了更好的参考。Objective To construct eukaryotic expression vector of human R-spondin 2(roof plate-specific spondin 2,RSPO2),express and purify fusion protein RSPO2-Fu-Fc,and detect the biological activity of RSPO2-Fu after resecting the Fc domain with 3C enzyme.Methods Based on bioinformatics method,the physical and chemical properties and structure of human RSPO2 protein were analyzed,and the truncated protein RSPO2-Fu which could exist stably in solution was screened.The gene encoding RSPO2-Fu protein was subcloned into pCMV-Fc vector,and then the plasmid pCMV-RSPO2-Fu-Fc was transiently transfected into HEK293F cells and expressed for 72 h.The fusion protein RSPO2-Fu-Fc was purified by Protein A chromatography column,the Fc domain was removed by 3C enzyme,and the RSPO2-Fu protein was further purified by using molecular sieve.Finally,the biological activity of RSPO2-Fu protein was detected by M50 Super 8×TOPFlash.Results Bioinformatics analysis showed that RSPO2 was a hydrophilic unstable protein with 243 amino acids,a relative molecular mass of 28300 and an isoelectric point of 9.42.The stability of RSPO2-Fu truncated protein in solution was greatly improved.The recombinant expression plasmid pCMV-RSPO2-Fu-Fc was constructed correctly as identified by colony PCR and sequencing,and RSPO2-Fu protein with a purity of 95%was obtained.TOPFlash results showed that the EC_(50)value of RSPO2-Fu was 1.5×10^(-12)mol/L.Conclusion After proper truncation of RSPO2 protein,stable expression of RSPO2-Fu protein can be obtained,which can obviously activate Wnt signaling pathway,and provides a better reference for RSPO2 related biological research in Wnt signaling pathway.

关 键 词：哺乳动物蛋白表达系统 重组人R-spondin2蛋白 荧光素酶报告基因 WNT信号通路 

分 类 号：Q51[生物学—生物化学]