海洋科学Issue(12)：22-28,7.DOI:10.11759/hykx20140223003
大黄鱼β-actin抗体的制备与组织表达谱分析
Expression and purification of β-actin antibody from Larim-ichthys crocea
摘要
Abstract
In order to evaluate the feasibility that theb-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 ana-lysed 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 pro-karyotic 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. Con-sequently, 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 pro-tein 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.关键词
大黄鱼(Larimichthys crocea)/beta-肌动蛋白/原核表达/抗体/组织表达谱Key words
Larimichthys crocea/beta-actin/prokaryotic expression/antibody/tissues expression分类
资源环境引用本文复制引用
董乐,姚丽梅,戴聪杰,张乐,黄苹苹,王建颖..大黄鱼β-actin抗体的制备与组织表达谱分析[J].海洋科学,2014,(12):22-28,7.基金项目
国家自然科学基金资助项目(31272679) （31272679）
福建省高校服务海西建设重点项目(A101) （A101）
福建省生物学省级重点学科建设项目(zdxk) （zdxk）
泉州市科技计划重点项目(2013Z126) （2013Z126）
