Studies On Key Genes Along Grow Axis From Cynoglossus Semilaevis Gǖnther: In Vitro Recombinant Expression And Roles In Growth And Reproduction Regulation

The in vitro prokaryotic expression systems of key endocrine genesincluding growth hormone (GH), Insulin-like growth factors (IGF-I andIGF-II) along the growth axis of Cynoglossus semilaevis Günther wereconstructed using molecular cloning, prokaryotic expression and westernblotting methods, the purified and biologically active recombinant proteinswere obtained; the obvious growth promotion effects of recombinantbovine GH and recombinant Cynoglossus semilaevis Günther IGF-I proteinon growth performance of Cynoglossus semilaevis Günther juveniles wereobserved through injection treatment, and serum GH and IGF-I levels,digestion enzyme levels were analyzed to clarify the possible underlyingmechanisms; finally, the possible roles and function mechanisms of GHand IGF-I in reproductive cycle regulation of Cynoglossus semilaevisGünther were revealed using ELISA, quantitative real-time PCR andhistological section methods, which were helpful for better understandingof the underlying endocrine mechanisms of Cynoglossus semilaevisGünther reproduction. 1. Prokaryotic expression and bioactivity analysis of GH, IGF-I andIGF-II from Cynoglossus semilaevis GütherPrimers were designed based on the full-length cDNA sequence of IGF-Iand IGF-II from Cynoglossus semilaevis Güther to clone the maturepeptide domains of IGF-I and IGF-II genes. The matured peptide of IGF-Iand IGF-II are both composed of70amino acids, including four functionaldomains B-C-A-D. The matured peptides fragments were subcloned intothe prokaryotic expression vector pET-28a to successfully constructrecombinant plasmid. Then the recombinant plasmid was transformed intoE. coli BL21(DE3) cell which can be induced by IPTG to produce aspecial fusion polypeptide containing His6at the N-terminus. TheSDS-PAGE analysis indicated that the obtained IGF-I and IGF-IIpolypeptides both with molecular weight of11.4K Da. The recombinantIGF-I protein proximately accounted for58.5%of the whole bacterialprotein post3-hour induction with IPTG, which meant1L of broth couldproduce40.7mg target protein based on the analysis of SigmaScansoftware. The recombinant IGF-II protein proximately accounted for43.7%of the whole bacterial protein post2-hour induction with IPTG. Westernblotting analysis indicated the two fusion polypeptides had the antigenicityto His6antibody. The IPTG-induced bacterial precipitation wasdenaturalize using6mol/L guanidine HCl, purified using Ni-NTA affinity chromatography and annealed by gradient dialysis in urea, then the purifiedrecombinant IGF-I and IGF-II protein with molecular weight of11.4KDwere obtained. The proliferation experiment showed recombinant IGF-Iand IGF-II proteins could significantly promote the proliferation of humanbreast cancer cells MDA231which verified their biological activities.Results from the present study could provide basic information andpractical methods for growth regulation of farmed Cynoglossus semilaevisGüther.A pair of primers was designed based on the full-length cDNA sequence ofGH from Cynoglossus semilaevis Güther to clone the mature peptidedomain of GH gene. The matured peptide from Cynoglossus semilaevisGüther is composed of183amino acids. The matured peptide fragment wassubcloned into the prokaryotic expression vector pET-28a to successfullyconstruct recombinant plasmid. Then the recombinant plasmid wastransformed into E. coli BL21(DE3) cell which can be induced by IPTG toproduce a special fusion polypeptide containing His6at the N-terminus.The SDS-PAGE analysis indicated that the obtained IGF-I polypeptide withmolecular weight of26KD. The recombinant GH protein proximatelyaccounted for41.5%of the whole bacterial protein post4-hour inductionwith IPTG based on the analysis of SigmaScan software. Western blottinganalysis indicated the fusion polypeptide had the antigenicity to His6 antibody. The IPTG-induced bacterial precipitation was denaturalize,purified and annealed, then the purified protein with molecular weight of26KD was obtained. ELISA for detection of recombinant GH proteinshowed the recombinant GH protein has ideal antigen activity. Theproliferation experiment showed recombinant GH protein could inhibit theproliferation of human breast cancer cells MDA231.2. Effects of exogenous recombinant GH and IGF-I on growthperformance of Cynoglossus semilaevis Güther juvenilesThe obvious growth promotion effects of recombinant bovine GH andrecombinant Cynoglossus semilaevis Günther IGF-I protein on growthperformance of Cynoglossus semilaevis Günther juveniles were observedthrough injection treatment, and serum GH and IGF-I levels, digestionenzyme levels were analyzed to clarify the possible mechanisms:recombinant bovine GH could promote the growth of Cynoglossussemilaevis Günther juveniles at dosage of10μg/Ind., the body weightgrowth rate was28.38%higher than that of control fish; recombinantCynoglossus semilaevis Günther IGF-I could obviously promote the growthperformance of Cynoglossus semilaevis Günther at dosage of2μg/Ind. and25μg/Ind., with the body weight growth rates were35.75%and50.91%(P＜0.05) higher than control fish respectively. The increased foodconversion rate, intestinal protease activity and intestinal amylase activity were observed in growth promoted experimental fish. Whereas, theendogenous serum GH and IGF-I levels did not significantly respond toexogenous GH and IGF-I treatment. The results showed the regulation ofgrowth performance of exogenous GH and IGF-I may depend on regulationof food conversion rate and digestion enzyme activity. The results could behelpful to better understand the function and mechanism of growth axis,also could provide useful materials for construction of practical regulationtechnique for growth of Cynoglossus semilaevis Günther.3. The roles and possible mechanisms of GH and IGF-I in ovarianmaturation of Cynoglossus semilaevis GüntherThe physiological roles of GH and IGF-I in ovarian maturation ofCynoglossus semilaevis Günther were investigated by combing thehistological sectioning, quantitative PCR detection and serum GH andIGF-I levels measurement. Results showed that pituitary GH mRNA levelincreased with ovary maturation and peaked at V stage (spawning period),thereafter, it significantly decreased to lower level (P＜0.05). Liver IGF-ImRNA level significantly decreased until ovary development attained stageⅣ, thereafter it increased and peaked after the spawning period (P＜0.05).Ovarian IGF-I mRNA level significantly increased with ovary developmentand peaked at stage Ⅳ(P＜0.05), but significantly decreased at stage Vand VI. Pituitary IGF-I mRNA level peaked at stage Ⅲ and did not varied obviously during the maturation cycle. The brain IGF-I mRNA levelincreased since stage Ⅲ and peaked at stage V.As for serum GH and IGF-I levels were concerned, the minimum serumGH level was found at stage Ⅲ, then significantly increased and peaked atstage V, which showed similar variation with pituitary GH mRNA levels.Serum IGF-I level achieved the minimum level at stage Ⅳ, and thensignificantly increased at stage V and peaked at stage Ⅵ. These resultsrevealed that the key growth regulators-GH and IGF-I also were involvedinto the reproduction regulation in Cynoglossus semilaevis Günther, thedetailed function and mechanisms need to be further studied.