CAJ | 학술논문

为表达和纯化尼罗罗非鱼(Oreochromis niloticus)cyp19a1a蛋白，本研究从尼罗罗非鱼卵巢提取总RNA，反转录获得cDNA模板后，利用设计的引物PCR扩增cyp19a1aORF区1200 bp片段，双酶切后连接到 pET-28a(＋)表达载体上，经酶切验证和 DNA 测序鉴定，证明成功构建了pET-28a-cyp19a1a原核表达载体。将表达载体转化到大肠杆菌(E.coli) BL21中，优化IPTG诱导浓度和诱导时间。结果显示，在0.5 mmol/L IPTG诱导8 h后，cyp19a1a重组蛋白大量表达，并以包涵体形式存在。通过 Ni2+-NTA 层析柱和切胶纯化，获得与预期片段大小一致的表达蛋白，并用Western blotting验证了纯化的蛋白为目的蛋白。本研究为制备cyp19a1a抗体和研究高温对cyp19a1a蛋白表达的影响提供了重要的基础。
위표체화순화니라라비어(Oreochromis niloticus)cyp19a1a단백，본연구종니라라비어란소제취총RNA，반전록획득cDNA모판후，이용설계적인물PCR확증cyp19a1aORF구1200 bp편단，쌍매절후련접도 pET-28a(＋)표체재체상，경매절험증화 DNA 측서감정，증명성공구건료pET-28a-cyp19a1a원핵표체재체。장표체재체전화도대장간균(E.coli) BL21중，우화IPTG유도농도화유도시간。결과현시，재0.5 mmol/L IPTG유도8 h후，cyp19a1a중조단백대량표체，병이포함체형식존재。통과 Ni2+-NTA 층석주화절효순화，획득여예기편단대소일치적표체단백，병용Western blotting험증료순화적단백위목적단백。본연구위제비cyp19a1a항체화연구고온대cyp19a1a단백표체적영향제공료중요적기출。
Cyp19a1a gene encodes aromatase, the key enzyme that converts androgens into estrogens. However little is known about the protein expression and purificationof this gene. In this study, the total RNA was extracted from the ovary of Nile tilapia and was then reverse-transcribed to cDNA. The 1221 bp ORF partial regionofcyp19a1a was amplified using RT-PCR, and the amplified fragments were purified for the following cloning. The amplified DNA fragments were ligated into pET-28a(+) expression vector for the construction of the prokaryotic expression vector pET-28a-cyp19a1a. The pET-28a-cyp19a1a vector was verified with restriction endonuclease digestion and DNA sequencing, and then transformed intoE.coli BL21. IPTG was applied to induce the expression of pET-28a-cyp19a1a recombinant proteins. In order to optimize the protein expression we tested the inducing effects of IPTG at concentrations from 0.1 to 1.0 mmol/L. The results showed that the expression of pET-28a-cyp19a1a recombinant proteins started at 2 h after the induction with 0.5 mmol/L IPTG. The expression reached the highest level at 8 h after the induction, and began to decrease at 10 h. Nonetheless the expression levels were not significantly different at various IPTG concentrations. Therefore the optimal induction conditions were determined to be 0.5 mmol/L IPTG for 8 h. The expressed recombinant proteins were mainly found in collected cells but not in the supernatant, which indicated that these proteins formed inclusion bodies. After the purification with Ni2+-NTA agarose gel chromatography column we obtained the products with the expected size (48 kDa). Next the products were further purified into the specific recombinant proteins using the KCl staining method. The concentration of purified protein was 0.82μg/μl. Western blotting results showed that the purified proteins can be detected by the His-tag antibody; hence they should be the target products. The successful expression and purification of the recombinant cyp19a1a protein would fundamentally improve the production of cyp19a1a antibody and provide insights into the effects of high-temperature on the sex differentiation in Nile tilapia.