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目的:克隆和表达G蛋白竞争性抑制肽(GCIP)基因,并研究其抗心肌肥大活性。方法:克隆GCIP基因,采用RTS500 系统表达,镍螯合亲和层析方法纯化;测定其对培养细胞[~3H]亮氨酸掺入量及总蛋白含量的影响,观察其抗心肌肥大的活性。结果:构建了pTVEX2.3MCS-GCIP表达载体,用RTS500系统成功表达了GCIP,RTS表达的GCIP占反应液总蛋白的2.43％,RTS系统表达的GCIP经镍柱纯化后,SDSPAGE显示均能得到一条分子量约8.5kDa的单一的条带,对GCIP的抗心肌肥大作用做了初步研究,结果显示GCIP 100μg/L、1mg/L和 10mg/L组[~3H]Leu掺入量和总蛋白含量已明显减少(P<0.01)。结论:成功构建了GCIP的表达载体pTVEX2.3MCS-GCIP,用RTS500系统表达GCIP蛋白,并经镍柱纯化,其表达量约为100mg/L,表达的GCIP在细胞模型具有抗心肌肥大活性。
AIM: To clone and express G protein competitive inhibitory peptide (GCIP) gene and study its anti-cardiac hypertrophy activity. Methods: The GCIP gene was cloned and expressed by RTS500 system and purified by nickel chelate affinity chromatography. The effect of levynyl - [3H] leucine incorporation and total protein content in cultured cells was assayed, and its anti - cardiac hypertrophy activity . Results: The pTVEX2.3MCS-GCIP expression vector was constructed and GCIP was successfully expressed in RTS500 system. The GCIP of RTS expression accounted for 2.43% of the total protein in the reaction solution. GCIP expressed by RTS system was purified by nickel column and SDSPAGE showed a The molecular weight of about 8.5kDa a single band on GCIP anti-cardiac hypertrophy done a preliminary study showed GCIP 100μg / L, 1mg / L and 10mg / L group [~ 3H] Leu incorporation and total protein content has been Significantly reduced (P <0.01). CONCLUSION: GCIP expression vector pTVEX2.3MCS-GCIP was successfully constructed. GCIP protein was expressed by RTS500 system and purified by nickel column. The expression level of GCIP was about 100 mg / L. The expressed GCIP had anti-cardiac hypertrophy activity in the cell model.