渔业科学进展Issue(4)：45-50,6.DOI:10.11758/yykxjz.20140407
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 Tilapiacyp19a1a Gene
摘要
Abstract
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.关键词
尼罗罗非鱼/cyp19a1a/原核表达/蛋白纯化Key words
Nile tilapia/Cyp19a1a/Prokaryotic expression/Protein purification分类
农业科技引用本文复制引用
王金,春根,赵燕,王慧,王艺雅,季相山..尼罗罗非鱼（Oreochromis niloticus）cyp19a1a原核表达与蛋白纯化[J].渔业科学进展,2014,(4):45-50,6.基金项目
山东省优秀中青年科学家科研奖励基金(BS2013NY002)和山东省现代农业产业技术体系共同资助。 （BS2013NY002）
