Recombinant Gonadotropin Of Turbot Scophthalmus Maximus By Eukaryotic Expression In Vitro

The availability of fish gonadotropins has traditionally relied on the extraction and purification of natural hormones from fish pituitary glands.Compared with natural purified hormones,the advantage of recombinant Gt H is that they can be produced continuously and do not need to rely on fish as a starting source.Turbot,as an important economic fish,cannot lay eggs naturally during artificial reproduction.In factory farming,it is necessary to regulate light and temperature and inject relevant hormones to promote the growth,maturation and ovulation of oocytes.At present,relevant hormones commonly used in the market have been used in actual production,and specific gonadotropins specifically for the breeding of turbot broodstock have not been developed.At the same time,many details of the function and mechanism of fish gonadotropins are still unclear.Therefore,this study took turbot as the research object and constructed a recombinant Pichia yeast strain to express turbot gonadotropin in vitro.The main results are as follows:1.Using turbot pituitary RNA as template,using RACE technology,successfully amplified turbot follicle stimulating hormone(FSHβ)c DNA full length,the gene sequence was submitted to NCBI,and the accession number was MT334574.After splicing the middle fragment,3’-RACE and 5’-RACE sequences,the 597 bp full-length c DNA sequence of turbot FSHβ gene was obtained.This sequence encodes 121 amino acids,including 5’non-coding region 117 bp and 3’non-coding region 117 bp.Analysis of its encoded amino acid sequence shows that its relative molecular weight is 12.96 Ku,the front end contains a signal peptide structure of 18 amino acids(1aa-18aa),and there are 3 potential phosphorylation sites and 1glycosylation site.2.Using turbot pituitary RNA as a template,using overlapping PCR technology,the αsubunit was connected to the two β subunits respectively,and the two fusion subunit sequences of CL and CF were cloned and digested with double enzymes(Eco R I,After Not I),the pPICZA plasmid was cloned in one step to obtain eukaryotic recombinant expression vectors: pPICZA-CL,pPICZA-CF.3.The eukaryotic recombinant expression vectors pPICZA-CL and pPICZA-CF were introduced into the Pichia pastoris GS115 strain using Quick & Easy Yeast Transformation Mix(TAKARA),and the best was obtained by optimizing the culture time and the methanol concentration of the inducerCultivation conditions: 30 ℃ 200 rpm,0.5% methanol is added every 24 h,the protein expression is the highest when cultured for 72 h.Expand the culture under optimal expression conditions,collect the culture supernatant for purification,and verify the successful expression of recombinant turbot gonadotropins(rCL,rCF)by SDS-PAGE and Western Blot verificationIn this study,the gonadotropin a subunit and the β subunit were successfully spliced,and the turbot gonadotropin dimer was successfully expressed using the eukaryotic expression system,which laid a foundation for the construction of an efficient in vitro recombinant expression platform for turbot gonadotropin Foundation,to provide technical support for the maturation and spawning of turbot oocytes.