Recombinant Expression, Purification and Functional Analysis of Half-Smooth Tongue Sole CSFR2 Protein

Abstract:

Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture species in the north coastal area of China. Male and female C. semilaevis exhibit distinct properties, yet their sex determination mechanism is complex and obscure. By genome and transcriptome sequencing of C. semilaevis, we identified a sex-related gene CSFR2, and we then carried out the cloning, expression in E. coli, and 1-step Ni-NTA-based purification to investigate the function of CSFR2. We injected the recombinant CSFR2 protein into C. 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 into E. 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 of Cyp19a and Foxl2. The results showed that the expression of Cyp19a and Foxl2 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 and Cyp19a, indicating that CSFR2 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.