人甲状旁腺素活性片段前体物Pro-Pro-hPTH(1-34)的制备及活性研究

目的制备出具有生物活性的人甲状旁腺素活性片段类似物,并对其活性进行考察。方法采用基因工程方法:大肠杆菌BL 21为宿主菌,pET 28a为表达载体,天门冬酰胺酶Ⅱ切除信号肽的C末端片段为融合伙伴,在融合伙伴和目的肽结合处引入Asp Pro酸敏感位点及二肽酶Ⅳ识别的N端Pro Pro位点,之后经诱导表达,酸水解使融合伙伴和目的肽分离。结果成功构建了hPTH(1 34)的融合表达体系,它能高效表达含hPTH(1 34)的融合蛋白,经纯化,得到多肽纯品,经鉴定,该多肽的分子量、氨基酸组成均与设计的分子相符,工程菌中用于编码该多肽的DNA片段其测序结果也与原设计相符,是表达该多肽的正确模板。该多肽在Parsons雏鸡分析及卵巢摘除大鼠模型试验中均显示显著活性。结论用基因工程方法成功地制备出人甲状旁腺素活性片段类似物,该产物具有显著生物活性及潜在的药用价值。

Research on preparation and activity of human parathyroid hormone fragment analogue Pro-Pro-hPTH(1-34)

Objective To prepare analogue of human parathyroid hormone fragment which possesses biological activity.Methods Gene engineering strategy was adopted taking E.coli BL-21 as the host bacterium,pET-28a as the expressing vector,C-terminal fragment of asparaginase II as the fusion partner whose signal peptide had been removed.An acid labile Asp-Pro site and a DPP Ⅳ recognizable N-terminal Pro-Pro-site were introduced between the fusion partner and the target peptide.The fusion protein was expressed after induction,and then separated in to the fusion partner and the target peptide through acid hydrolysis.Results The fusion expressing system of hPTH(1-34) was successfully constructed.Using this system,the fusion protein containing hPTH(1-34) was efficiently expressed.Pure peptide was obtained after separation and purification.Its molecular weight and amino acid composition fitted with the peptide molecular originally designed;the DNA sequence analysis of the bacterium expressing this peptide gave a completely same sequence with the DNA fragment originally designed for coding Pro-Pro-hPTH(1-34).It showed biological activity in Parsons's Chicken assay and pharmacological activity in ovariectomized rat model.Conclusions An analogue of human parathyroid hormone fragment was successfully prepared using gene engineering strategy,which showed significant biological activity and potential pharmaceutical value.