中文摘要：

为探讨大黄鱼（Larimichthyscrocea）β-actin基因化cActb）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Westernblotting验证；原核表达结果表明，IPTG可诱导一个分子量约45ku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／LIPTG、200r／min诱导表达5h。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Westemblotting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Westernblotting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。

英文摘要：

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 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/rain 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 ofL. 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.