| 郭寰宇 董文博 曲晓波 史文婷 郭焱.鹿茸富脯氨酸多肽-APRP基因融合表达载体的构建及表达[J].,2016,16(23):4419-4422 |
| 鹿茸富脯氨酸多肽-APRP基因融合表达载体的构建及表达 |
| Construction of Fusion Expression Vector and Inducible Expression of theAntler Proline-rich Polypeptide-APRP |
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| DOI： |
| 中文关键词: APRP GST-APRP 融合表达载体 诱导表达 |
| 英文关键词: APRP GST-APRP Fusion expression vector Inducible expression |
| 基金项目:十二五" 国家科技支撑项目（2011BAI03B02-2） |
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| 中文摘要: |
| 目的：构建鹿茸富脯氨酸多肽（APRP）基因融合表达载体，并诱导表达融合蛋白GST-APRP。方法：以鹿茸顶端组织总cDNA
为模板，PCR 方法扩增目的片段，连接pMDTM18-T 载体，转化DH5琢进行TA克隆。酶切与测序鉴定合格后，将目的片段
与pGEX-6P-1 质粒连接，完成pGEX-6P-1-APRP融合表达载体的构建。利用原核表达系统进行初步诱导表达。SDS-PAGE 蛋白电
泳检测蛋白表达情况。结果：PCR 成功扩增出目的基因片段，目的基因片段大小为216 bp。提取表达载体质粒经酶切及测序分析
证实pGEX-6P-1-APRR 融合表达载体构建成功。利用终浓度为0.5 mMIPTG 诱导，融合蛋白成功表达，分子量为35 kDa。结论：目
前利用基因工程技术获得特定的鹿茸多肽的报道很少。本研究结果表明，利用原核表达系统可在体外对鹿茸富脯氨酸多肽基因
（APRP）进行融合表达，并且融合蛋白GST-APRP 以可溶形式表达，有利于进一步研究APRP。这也为研究其他鹿茸多肽单体成分
提供重要参考。 |
| 英文摘要: |
| Objective:To construct the fusion expression vector of antler proline-rich polypeptide (APRP), and induce the expression
of fusion protein GST-APRP.Methods:The APRP segment was amplified by PCR technology with total cDNA template of velvet
antler top organization. The product of amplification was linked with pMDTM18-T vector, and the plasmid was transformed into
DH5琢to accomplish TA clone. The fusion expression vector pGEX-6P-1-APRP was constructed by linking the target segment with
pGEX-6P-1 vector after the correct identification of enzyme digestion and sequencing. Prokaryotic expression system was used to induce
the expression of protein preliminarily, and expression was detect by SDS-PAGE protein electrophoresis.Results:We amplified the
APRP gene fragment by PCR, the length of it was 216bp, and constructed the fusion expression vector pGEX-6P-1-APRR successfully.
We obtained the fusion protein whose molecular weight was 35 kDa by using 0.5 mMIPTG of final concentration.Conclusion:The reports'
quantity of obtaining specific velvet antler polypeptides by genetic engineering technology is rare currently. This study results show
that we can induce the fusion expression of antler proline-rich polypeptide(APRP) in vitro, and GST-APRP fusion protein expressed with
soluble form, which is helpful for further research of APRP. These efforts are also providing a valuable reference for the researching of
other monomer composition of velvet antler polypeptides. |
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