半滑舌鳎(Cynoglossus semilaevis)CSFR2基因的重组表达及其蛋白纯化与功能分析

摘要

通过对半滑舌鳎(Cynoglossus semilaevis)进行全基因组及转录组测序,预测了一个半滑舌鳎性别相关基因CSFR2,目前已对该基因进行了克隆和表达分析.为进一步研究CSFR2基因的功能,本研究从蛋白层面入手,构建了原核表达载体pET-32a-CSFR2,转化到大肠杆菌E. coli进行诱导表达,用His Trap进行蛋白纯化,SDS-PAGE电泳检测诱导及纯化产物,并将表达纯化的CSFR2蛋白注射1龄半滑舌鳎精巢.由于CSFR2是一个性别相关基因,因此无法通过免疫反应来直接检测其蛋白活性,本研究通过不同时间点取样检测半滑舌鳎其他性别相关基因Cyp19a和Foxl2的表达量变化,来间接反应 CSFR2 蛋白的活性及功能.实验结果显示,重组蛋白能够进入到活体细胞内并发挥一定作用,在6–72 h内对性别相关基因Cyp19a和Foxl2的表达有上调作用,说明CSFR2蛋白具有生物活性,并能调节其他性别相关基因的表达量,间接促进雄激素转化为雌激素.本研究进一步了解 CSFR2 基因在半滑舌鳎性别决定与分化中的作用,可为半滑舌鳎性别控制提供理论依据,在人工诱导鱼类性逆转方面有重要应用价值.此外,本研究为蛋白活性验证提供了一种新方法.%Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture species in the north coastal area of China. Male and femaleC. semilaevis exhibit distinct properties, yet their sex determination mechanism is complex and obscure. By genome and transcriptome sequencing ofC. semilaevis, we identified a sex-related geneCSFR2, and we then carried out the cloning, expression inE. coli, and 1-step Ni-NTA-based purification to investigate the function ofCSFR2. We injected the recombinant CSFR2 protein intoC. semilaevis and quantified the amounts of two genes presumably to be affected at the transcriptional level using qPCR. The marker genes were positively affected during the first 72 h following injection. A prokaryotic expression plasmid pET-32a-CSFR2 was constructed transformed intoE. coli to produce the fusion protein. HisTrap HP was used for protein purification, and SDS-PAGE electrophoresis was used for fusion protein detection. We injected the fusion protein with liposome into fish, and tested the expression ofCyp19a andFoxl2. The results showed that the expression ofCyp19a andFoxl2 was significantly up-regulated between 6 to 72 h and then returned to the basal level at 72 h after injection. Being a sex-related gene, the activity of the CSFR2 fusion protein could not be directly detected using immunoreaction. We hence examined the activities of other sex-related genes, which could reflect the activity of CSFR2. The results showed that the recombinant CSFR2 protein up-regulated the expression of female-related genes,Foxl2 andCyp19a, indicating thatCSFR2 played a role in sex differentiation by regulating the expression of other sex-related genes. Our study proposed a new strategy in the gene function study, and provided fundamental information for the artificial induction of fish sex reversal.