人源Dickkopf1原核表达质粒的构建及表达产物初步鉴定

Construction of Human Dickkopf1 Prokaryon Expression Plasmid and Identification the Product of Expression

目的构建人源dickkopf1(dkk1)基因的原核表达质粒,表达、纯化、鉴定其表达产物。方法自足底皮肤组织提取成纤维细胞,抽提mRNA,RT-PCR获得人源dkk1基因片断,构建重组质粒PQE30-dkk1;转化至大肠杆菌内,诱导表达,超声裂菌、镍琼脂糖凝胶层析纯化表达产物,SDS-PAGE电泳、WesternBlot鉴定表达产物。结果RT-PCR获得片断与预期结果相符,双酶切鉴定、测序鉴定证实重组质粒PQE30-dkk1构建成功;SDS-PAGE电泳、抗Dkk1抗体WesternBlot鉴定表达产物为Dkk1蛋白。结论成功构建了人源dkk1的原核表达质粒,并表达、纯化、鉴定了该基因的原核表达产物,为下一步的Dkk1蛋白对皮肤黑素细胞的生物学活性研究奠定基础。

Objective To clone,express,purify and verify human Dickkopfl （ Dkkl ）. Methods Fibroblasts from metatarsus were cultured,and mRNA was harvested from the fifth generation, The dkkl gene was acquired via RT-PCR and cloned in PQE30 plasmid containing the 6His tag to construct the recombinant plasmid PQE30-dkk1. The E. coli was amplified and induced via IPTG after the PQE30-dkk1 translated in. The E, coli was split ultrasound, The product of expression was purified with NI-agarose chromatography and verified via SDS-PAGE and Western Blot. Results The product of RT-PCR was coincide with what we preconceived, Double restriction enzyme cutting and sequence-analysis proved the recombinant plasmid to be PQE30-DKK1. The product of expression was verified properly via SDS-PAGE electrophoresis and Western Blot with special anti-DKK1 antibody, Condusion The successful cloning of dkkl gene and expression of Dkkl protein lay a basis for studying the effects on the biologic activity of melanocyte.