CAJ | 학술논문

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。
위탐토대황어(Larimichthys crocea)β-actin 기인(LcActb)작위내삼기인진행상관연구적가행성，종전록화번역수평연구기조직표체보，작자이대황어간장위재료，확증 LcActb 적개방열독광서렬，병장기아극륭지원핵표체재체 pET32a(+)。매절、측서결과표명， pET32a-LcActb 구건성공。장pET32a-LcActb전화도대장간균BL21(DE3)중진행우화표체，표체단백경순화후진행Western blotting험증；원핵표체결과표명， IPTG 가유도일개분자량약45 ku적특이단백，차주요이포함체적형식존재，우화표체조건위：28℃、0.4mmol/L IPTG、200 r/min유도표체5 h。용표체단백작위항원，면역신서란백토제비항LcActb적다극륭항체；용순화적다항진행Western blotting적결과표명，획득료특이성적LcActb항체；통과실시형광정량RT-PCR 검측대황어다충조직mRNA적표체，기차，제비료다충조직균장단백，사용순화적항체진행Western blotting분석，결과현시， LcActb재대황어성체각조직중전록화번역수평적표체차이균불현저，가주위연구대황어성체조직중기타기인표체화번역수평적내삼표준。
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.