| 摘要: |
| 【目的】构建pTAT-bSox2-9R融合表达载体，并对其进行原核表达研究，为利用TAT和多聚精氨酸(9R)的跨膜作用实现转录因子蛋白直接重编程牛体细胞提供科学资料。【方法】以GenBank上公布的牛Sox2基因编码区序列为基础，设计并合成含酶切位点、跨膜转导肽TAT和9R的引物；利用PCR扩增方法获得包括Sox2、TAT和9R的重组DNA序列，并将其克隆到空质粒pTAT-HA上，从而构建pTAT-HA-bSox2-9R重组表达载体；将经酶切鉴定和测序验证后的重组表达载体转化大肠杆菌，在不同IPTG浓度和诱导时间等条件下进行诱导表达，对表达产物进行SDS-PAGE和Western blot分析。【结果】成功获得bSox2-9R重组序列,成功构建重组质粒pTAT-bSox2-9R；pTAT-bSox2-9R在0.6 mmo/L的IPTG于37 ℃诱导4 h的条件下表达目的蛋白约36 ku，经Western blot验证为目的蛋白。【结论】成功获得了pTAT-bSox2-9R融合蛋白，为多聚精氨酸蛋白转导域（PTD）介导的转录因子蛋白直接重编程黄牛体细胞以获得诱导多潜能干细胞的研究积累了原始资料。 |
| 关键词: 黄牛 体细胞重编程 TAT Sox2 多聚精氨酸（9R） 原核表达 |
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| 基金项目:国家自然科学基金项目(31000655)；西北农林科技大学博士启动基金项目（BSJJ2010049） |
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| Construction and prokaryotic expression of TAT-bSox2-9R fusion expression vector |
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| Abstract: |
| 【Objective】The objective of this study was to construct and express the TAT-bSox2-9R fusion vector,which would improve the formation of safe induced pluripotent stem cells using TAT and Poly Arg (9R) peptides.【Method】According to the available coding region of bovine Sox2 gene in GenBank database,the specific primers including enzyme recognizing site,TAT and 9R sequences were designed to amplify and obtain the recombinant DNA sequence including Sox2 gene coding region,TAT and 9R.Then,the recombinant DNA sequence was cloned to vector pTAT-HA to form recombinant vector pTAT-HA-bSox2-9R.After being identified by enzyme digestion analysis and DNA sequencing,the correct recombinant vector pTAT-HA-bSox2-9R was induced to express products under different concentrations of IPTG and different stages.Finally,the expressed products were identified via Western blot.【Result】Bovine Sox2 gene was successfully obtained,and the TAT-bSox2-9R fusion expression vector was successfully constructed.Then the vector was transformed into Escherichia coli BL21 (DE3) cells for expression.IPTG induction of the TAT-bSox2-9R expression was optimized when being treated with 0.6 mmol/L IPTG for 4 hours at 37 ℃.The expressed protein was 36 ku and was confirmed by Western blot.【Conclusion】This study successfully constructed the pTAT-bSox2-9R fusion expression vector and expressed the fusion protein TAT-bSox2-9R,which would provide useful material and information for reprogramming bovine somatic cell into iPS and promoting the cattle industry. |
| Key words: bovine somatic cell reprogramming TAT peptide Sox2 gene(9R) Poly Arg prokaryotic expression |
