尼罗罗非鱼(Oreochromis niloticus) cyp19a1a原核表达与蛋白纯化

The Prokaryotic Expression and the Protein Purification of Nile Tilapia cyp19a1a Gene

中文关键词: 尼罗罗非鱼  cyp19a1a  原核表达  蛋白纯化

英文关键词: Nile tilapia  Cyp19a1a  Prokaryotic expression  Protein purification

中文摘要:

      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cyp19a1a ORF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。

英文摘要:

      Cyp19a1a gene encodes aromatase, the key enzyme that converts androgens into estrogens. However little is known about the protein expression and purification of 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 region of cyp19a1a 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 into E.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 0.82 μg/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.