Reorganization Of Shrimp Breeding And Immune-related Functions Gene Expression And Immunohistochemistry Studies

Recombinant expression is an important technology to study on the genefunction. Base on the earlier research, we chose transcription factors of immunesignal pathway, Relish and Dorsal, and ovary development related genes, CASand SSCRF, as research objects. In this study, we successfully expressed thetarget recombinant proteins by optimizing the prokaryotic expression system andobtained purified proteins. Subsequently, we prepared rabbit anti-Relish, rabbitanti-Dorsal, mouse anti-CAS and mouse anti-SSCRF polyclonal antibodies. Also,The distributions of CAS and SSCRF in gonad of shrimp were analysed byimmuno-histochemical analysis.Fusion proteins, His-Relish,His-Dorsal,GST-CAS and GST-SSCRF weresuccessfully expressed in E. coli, which were purified by His Trap FF crude Kitand MagneGST Protein Purification System. Their molecuar weight were30kDa,39kDa,39kDa,44kDa, respectively.Antiserums of rabbit anti-His-Relish and His-Dorsal, mouse anti-GST-CASand GST-SSCRF were obtained and identified with Western Blot, respectively.Subsequently, immuno-histochemical analysis showed that:1. CAS protein existed both in the cytoplasm and nucleus of ovary,which suggested that CAS might be a carrier protein betweennucleus and cytoplasm. Combined with the data that the abnormallydeveloped ovaries of triploid shrimp had no expression of CAS, weinferred that CAS might be one of the key genes which areresponsible for the ovary development of shrimp.2. SSCRF protein existed both in the testis of diploid shrimp and ovaryof triploid shrimp, which suggested that SSCRF might influence theovary development of shrimp.The results have established a firm basis for further dissection on mechanisms of immune system and ovary development.