人甲状旁腺素N-端34肽基因的合成、克隆和融合表达

Synthesis,Cloning and Fusion Expression of Gene Encoding 34 Peptides at N-termi nus of Human Parathyroid Hormone

目的构建重组人甲状旁腺素N-端34(PTH34)肽的大肠杆菌融合表达菌株,并对其表达产物进行检测。方法选用大肠杆菌偏爱密码子,设计优化的人甲状旁腺素N-端34肽基因序列,通过6个DNA片段分段合成、片段连接、PCR扩增,经TA克隆到pMD18载体中,经测序验证,构建GST融合表达载体。结果经SDS-PAGE检测PTH(1-34)肽与GST蛋白的融合表达,经Westernblot分析具有免疫活性,通过GST亲和层析和Xa因子酶切,利用小分子蛋白电泳可检测到PTH(1-34)肽。结论已获得了能表达GST-PTH(1-34)融合蛋白的菌株。表达产物具有免疫活性,且能被Xa因子消化得到PTH(1-34)肽。

Objective To construct a recombinant E.coli strain expressing the 34 peptides at N-te rminus of human parathyroid hormone (PTH34) and identify the expressed product. Methods Design an optimal gene sequence encoding the PTH34 using E.coli-preferred c odons and synthesize 6 DNA fragments separately.Link the fragments with ligase,a mplify by PCR and clone into pMD18 vector by T-A cloning.Identify the recombina nt plasmid by DNA sequencing and transform to E.coli for GST fusion expressi on. Results The fusion expression of PTH(1-34) and GST protein was proved by SDS-PAGE.West ern blot showed immunological activity of expressed product.After the fusion pro tein was purified by GTST affinity chromatography and digested with Xa factor,PT H(1-34) was revealed by small molecule protein electrophoresis. Conclusion A recombinant E.coli strain expressing GST-PTH(1-34) was successfully cons tructed.The expressed product showed immunological activity and might be digeste d into PTH(1-34) with Xa factor.