重组人糖蛋白激素β5/α2融合蛋白在CHO-S细胞中的表达纯化及功能活性分析

Expression,purification,and functional verification of recombinant human glycoprotein hormone beta 5/alpha 2 fusion protein in CHO-S cells

目的在悬浮中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO-S)中分泌表达、纯化重组hCGH-CTP融合蛋白,验证其对3T3-L1成熟脂肪细胞脂质积累的影响。方法构建CTP连接肽融合人糖蛋白激素β5/α2重组蛋白表达载体pcDNA3.1-rhCGH-CTP,将其瞬时转染CHO-S悬浮细胞中,大量表达纯化并验证rhCGH-CTP蛋白生物学活性;通过干预3T3-L1成熟脂肪细胞24 h,观察细胞内甘油三酯(TG)水平的变化。结果Western blot结果显示,rhCGH-CTP蛋白在CHO-S细胞中成功表达,表达量可达715.4 mg·L^(-1);用AKTA pure蛋白纯化系统纯化蛋白,SDS-PAGE方法鉴定纯化出的蛋白纯度较高可达90%。此外,在高表达TSHR基因的成熟脂肪细胞3T3-L1中,利用ELISA试剂盒测定不同浓度rhCGH-CTP蛋白干预后胞内cAMP含量明显升高,说明rhCGH-CTP蛋白具有生物活性;油红O染色结果发现,与对照组相比,不同浓度rhCGH-CTP蛋白干预组的成熟脂肪细胞中TG含量明显降低(P<0.05)。结论成功表达并纯化了rhCGH-CTP融合蛋白,其具有良好的生物学活性并能有效降低TG,该研究为后续深入揭示CGH蛋白的生理作用及在临床实践中的潜在应用提供了重要基础。

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells(CHO-S),and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes.Methods The recombinant protein expression vector(pcDNA3.1-rhCGH-CTP)was constructed,achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker.The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified,and the protein biological activity was verified.Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride(TG)level.Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells,and the yield was up to 715.4 mg·L-1.The secreted protein was purified by AKTA pure system with higher purity that was up to 90%as identified by SDS-PAGE.In addition,the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit,indicating that rhCGH-CTP protein had biological activity.Oil red O staining showed that compared with the control group,the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased(P<0.05).Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity,and effectively reduce TG.This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.