N端延伸的人肠生长激素类似物基因的合成、克隆及融合表达

Synthesis,Cloning and Fusion Expression of N-Extended Human Gut Growth Hormone Analogue Gene in Escherichia coli

目的:合成、克隆及融合表达N端延伸2个Pro,并由Gly取代N端第2个氨基酸Ala的人肠生长激素类似物PP-h[Gly2]GLP-2,初步测定其促肠生长的生物学活性。方法:合成长度分别为53,54及53个碱基的三段寡核苷酸,以中段寡核苷酸为模板,两端寡核苷酸为引物,经PCR合成PP-h[Gly2]GLP-2基因,克隆于pUC18载体。经测序正确后,将该基因与融合表达载体pED连接构建融合表达质粒pEDGLP-2,转化E.coliBL21(DE3),进行诱导表达,用SDS-PAGE分析。融合蛋白经分离纯化和酸切割后,分离获得PP-h[Gly2]GLP-2,用电喷雾质谱法测定其相对分子质量,并对雄性SD大鼠皮下给药14 d,初步测定其促肠生长活性。结果:诱导后融合蛋白表达量占菌体可溶性总蛋白的40%以上。电喷雾质谱法测得:经酸切割获得的PP-h[Gly2]GLP-2的相对分子质量为3 947.97 Da,与预测值3 947.38 Da基本一致。初步动物实验结果显示,0.5 mg/(kg.d)剂量组小肠增重百分比为41.65%,与PBS组相比具有显著差异。结论:制备获得的人肠生长激素类似物PP-h[Gly2]GLP-2,与设计的相对分子质量一致,有明显的促肠生长活性。

AIM:To construct N-extended human gut growth hormone GLP-2 analogue PP-hGLP-2 and to study its intestinotrophic activities in vivo.METHODS:Three segments with 53,54 and 53 bases were synthesized to construct PP-hGLP-2 gene.The entire gene of 124 bp was obtained by PCR and cloned into pUC18.After sequencing,the PP-hGLP-2 gene was inserted into fusion expression vector pED which contains ansB-C gene.The recombinant plasmid pEDG-2 was used to transform E.coli BL21(DE3).The expression of the fusion protein was detected by 15% SDS-PAGE after induction.PP-hGLP-2 was obtained by acid cleaving and examined by ESI-MS.The intestinotrophic properties of PP-hGLP-2 in vivo was studied in male SD rats at the dose of 0.5 mg/(kg·d) for 14 days by sc.RESULTS:Examined by SDS-PAGE and gel scanning,the expression level of fusion protein was over 40% in the inclusion bodies.The molecular weight of PP-h[Gly2]GLP-2 was 3 947.97 Da,near the forecast data 3 947.38 Da.The weight of small intestine of PP-hGLP-2 group raised 41.65% compared with that of PBS group.CONCLUSION:The N-extended human gut growth hormone GLP-2 analogue PP-hGLP-2 gene was synthesized and cloned successfully and the expression product exhibited significant intestinotrophic activity.