| 摘要: |
| 【目的】克隆猪淋巴结双链RNA激活的蛋白激酶（PKR）基因，并在体外初步表达，为研究流感病毒感染时猪PKR与P58IPK在体内外的相互作用奠定基础。【方法】用Trizol法从猪淋巴结中提取总RNA，RT-PCR法扩增猪PKR基因的完整编码区，将该片段连接到原核表达载体pGEX-4T-1和真核表达载体pDsRed1-N1中，采用SDS-PAGE检测原核表达载体pGEX-PKR在大肠杆菌BL21中的表达情况，用脂质体转染法将真核表达载体pDsRed1-PKR转入猪脐静脉血管内皮细胞系（SUVECs），Western blot检测其在细胞中的表达。【结果】PCR扩增得到了1 754 bp的DNA片段，与预期结果一致；原核表达载体pGEX-PKR和真核表达载体pDsRed1-PKR经双酶切和核酸测序分析，表明载体构建成功；SDS-PAGE检测结果表明，pGEX-PKR重组质粒在大肠杆菌中成功表达；Western blot检测到pDsRed1-PKR重组质粒在SUVECs中成功表达。【结论】成功克隆了猪淋巴结PKR基因，并在体外初步表达成功。 |
| 关键词: 猪PKR基因 克隆 原核表达 真核表达 |
| DOI： |
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| 基金项目:国家自然科学基金项目(30270342，31072115) |
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| Cloning and vector construction of porcine lymph node PKR gene and its primary expression |
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| Abstract: |
| 【Objective】Porcine lymph node PKR gene was cloned and expressed primarily in vitro to lay a basis for investigating the interactions of PKR with P58IPK during infection of influenza virus.【Method】Total RNA from porcine lymph node were extracted using Trizol,and the intact ORF of porcine PKR was obtained by RT-PCR.Porcine PKR was cloned into the prokaryotic expression vector pGEX-4T-1 and eukaryotic expression vector pDsRed1-N1.The expression product of prokaryotic vector pGEX-PKR was detected by SDS-PAGE.Eukaryotic expression vector pDsRed1-PKR was transfected to SUVECs by lipofectamine 2000 and red fluorescent protein was detected by Western blot.【Result】Results showed that both prokaryotic expression vector and eukaryotic expression vector were constructed successfully.pGEX-PKR was expressed in E.coli BL21 which was confirmed by SDS-PAGE.PKR gene was successfully expressed in SUVECs which was confirmed by Red marker and Western blot.【Conclusion】The porcine lymph node PKR gene was cloned and expressed primarily in vitro. |
| Key words: PKR gene cloning prokaryotic expression eukaryotic expression |
