Molecular cloning and in vitro expression of growth hormone gene from Platichthys stellatus

The full-length cDNA encoding growth hormone(GH)was isolated from the pituitary of Platichthys stellatus using RACE(rapid amplification of cDNA ends)method.Results showed that Platichthys stellatus GH cDNA sequence is 957bp in length and encodes 204 amino acids.The highest amino acid identity of Platichthys stellatus GH was 73.0% with Paralichthys olivaceus.Phylogenetic analysis indicated that Platichthys stellatus GH was clustered with other pleuronectiformes and perciformes species.The tissue expression patterns of GH mRNA in different tissues of female and male adults were analyzed by the quantitative real-time PCR.GH mRNA showed the highest expression level in the pituitary of both sexes with lower levels in the brain,gonad,liver,stomach and muscle.The expression levels of GH mRNA in the female stomach and muscle were significantly higher than those of males(P<0.05),which implied that it might be involved in sexually dimorphic growth pattern through paracrine and autocrine pathways.The GH/pET28a recombinant plasmid was successfully constructed and highly expressed in E.coli BL21(DE3)after being induced by IPTG with special fusion polypeptides containing His6 at their N-terminus.The obtained recombinant GH polypeptide was expressed in form of inclusion bodies with molecular weight of 24.9 ku and had the antigenicity to His6 antibody by western blotting analysis.The inclusion bodies were denaturalized using 6mol/L guanidine HCl,purified using Ni²⁺-NTA affinity chromatography and annealed by gradient dialysis in urea,and the verified recombinant GH protein was obtained.The effect of recombinant GH protein on proliferation of human embryonic kidney cells HEK293T was tested,and the results showed that significant inhibition was only found when protein concentration was higher than 5.4 μg/mL.Results from the present study could provide basic knowledge for molecular-level and protein-level growth regulation mechanism study of Platichthys stellatus.