| 摘要: |
| 【目的】克隆山羊甲状旁腺相关肽（Parathyroid hormone related protein，PTHrP）基因，并进行原核表达，获得具有生物学活性的融合蛋白。【方法】无菌采集山羊乳腺组织，采用TRIZOL法提取总RNA，RT-PCR克隆山羊PTHrP基因全CDs区。将山羊PTHrP基因全CDs区插入pET-32a(+)表达质粒构建pET-PTHrP质粒，经酶切和测序鉴定后，将其转化E.coli BL21(DE3)菌株，经2 mmol/L IPTG、37 ℃诱导表达8 h后，进行SDS-PAGE和Western blotting分析。【结果】获得了山羊PTHrP基因全CDs区，长534 bp (GenBank登录号GU573787)；酶切和测序显示，原核表达载体pET-PTHrP构建成功；SDS-PAGE和Western blotting分析表明，在E.coli BL21(DE3)中成功诱导表达了pET-PTHrP融合蛋白。【结论】克隆了山羊PTHrP基因，获得了PTHrP融合蛋白。 |
| 关键词: PTHrP基因 原核表达 蛋白纯化 山羊 |
| DOI： |
| 分类号: |
| 基金项目:西北农林科技大学科研启动基金项目(08080112)；农业部公益性行业科研专项(3-45) |
|
| Cloning of Capra hircus PTHrP and its prokaryotic expression in Escherichia coli |
|
|
| Abstract: |
| 【Objective】This experiment was conducted to study Capra hircus parathyroid hormone related protein PTHrPgene expression in E.coli under the condition of cloning PTHrPgene complete CDs sequence.【Method】Total RNA was isolated from C.hircus mammary gland tissues.The CDs sequnence of PTHrP gene were amplified by RT-PCR.CDs of PTHrP was inserted to the prokaryotic expression vector pET-32a(+) and expressed(2 mmol/L IPTG for 8 h at 37 ℃)in E.coli BL21(DE3) host cells.The results were analysed by SDS-PAGE and Western boltting.【Result】The C.hircus PTHrP gene complete CDs was 534 bp.Restriction enzyme mapping and sequencing showed that prokaryotic expression vector was constructed successfully.SDS PAGE and Western blotting showed that fusion protein (36.5 ku) was induced in E.coli BL21(DE3) and purified.【Conclusion】 C.hircus PTHrP gene was cloned and expressed in this study,which laid a foundation for preparing polyclonal antibody and further studying on its function. |
| Key words: PTHrP gene prokaryotic expression protein purification Capra hircus |
