| 摘要: |
| 为探讨大黄鱼(Larimichthys crocea)β-actin 基因(LcActb)作为内参基因进行相关研究的可行性,从转录和翻译水平研究其组织表达谱, 作者以大黄鱼肝脏为材料, 扩增LcActb的开放阅读框序列, 并将其亚克隆至原核表达载体pET32a(+) 。酶切、测序结果表明, pET32a-LcActb构建成功。将pET32a-LcActb转化到大肠杆菌BL21(DE3) 中进行优化表达, 表达蛋白经纯化后进行Western blotting验证; 原核表达结果表明, IPTG 可诱导一个分子量约45 ku 的特异蛋白, 且主要以包涵体的形式存在,优化表达条件为: 28℃、0.4mmol/L IPTG、200 r/min 诱导表达5 h。用表达蛋白作为抗原, 免疫新西兰白兔制备抗LcActb的多克隆抗体; 用纯化的多抗进行Western blotting 的结果表明, 获得了特异性的LcActb抗体; 通过实时荧光定量RT-PCR 检测大黄鱼多种组织mRNA 的表达, 其次, 制备了多种组织匀浆蛋白, 使用纯化的抗体进行Western blotting 分析, 结果显示, LcActb在大黄鱼成体各组织中转录和翻译水平的表达差异均不显著, 可做为研究大黄鱼成体组织中其他基因表达和翻译水平的内参标准。 |
| 关键词: 大黄鱼(Larimichthys crocea) beta-肌动蛋白 原核表达 抗体 组织表达谱 |
| DOI：10.11759/hykx20140223003 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目(31272679); 福建省高校服务海西建设重点项目(A101); 福建省生物学省级重点学科建设项目(zdxk); 泉州市科技计划重点项目(2013Z126) |
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| Expression and purification of β-actin antibody from Larimichthys crocea |
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| Abstract: |
| In order to evaluate the feasibility that the β-actin gene of Large yellow croaker (Larimichthys crocea), named as LcActb, acts as internal reference gene for the relavent researchs, the expression level of LcActb was analysed in different tissues. LcActb was isolated from the total RNA of livers from L. crocea by RT-PCR with gene specific primers designed according to the sequence of open reading frame of published data. Furthermore, a prokaryotic expression vector pET32a-LcActb was constructed and transformed into E. coli BL21 (DE3). After induced by IPTG, a molecular mass of fusion protein close to 45 ku was produced, and the best expression was induced by 0.4 mmol/L IPTG at 28 ℃, 200 r/min for 5 h. The fusion protein was purified and examined by SDS-PAGE and western blotting analysis. LcActb proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blotting analysis that polyclonal antibodies against LcActb had high specificity. The relative expression levels of LcActb mRNA in L. crocea were determined by fluorescent Real-time RT-PCR. LcActb was steadily expressed in different tissues of L. crocea. Meanwhile, the expression of LcActb protein was also analyzed using the purified antibodies through Western blotting. It was found that LcActb was steadily expressed in the detected tissues. So this LcActb gene was suitable as an internal control for analysis of functional gene in L. crocea. |
| Key words: Larimichthys crocea beta- actin prokaryotic expression antibody tissues expression |
