Expression Study Of Grass Carp Growth Hormone Gene In Pichia Pastoris

Grass carp growth hormone (CGH) gene was correctly inserted into pGEX-4T-l plasmid in order to construct the expression plasmid pGCGH. The recombinant plasmid was transformed into E.coli TOP10F' competent cells, the positive clone was screened by colony PCR and plasmid PCR and the recombinant plasmid was identified by BamH I and EcoRI digestion. Fusion protein expression was induced by IPTG and expression situation was assayed by RT-PCR and SDS-PAGE. The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit. The result of RT-PCR showed that the recombinant plasmid could be transcripted correctly in TOP 10F' . A new protein band was found in SDS-PAGE with molecular mass of about 50 KDa which is consisted of a 23.6 KDa protein deduced from the CGH gene sequence and GST (26 KDa). The titer of rat anti-CGH serum from immunized rat was 1: 128,000.A new pair of primers of CGH gene was designed which contained aEcoRI site in sense primer and a Not I site in antisense primer, and a new CGH gene fragment was obtained by amplification which didn't contain signal peptidc and intron. The new fragment was inserted into pPIC9K plasmid which contain methanol promoter and yeast signal peptide toconstruct expression plasmid pPIC9K - CGH, and the plasmid was transformed into Pichia Pastoris by electroporation. Four high copy positive clones were screened and purified by culturing on MD plate , MM plate and YPD-G418 plat and culture of the four clones was purified by saturation ammonium sulfate followed by SDS-PAGE assay, and a band of about 23.6KD was found which showed that CGH gene was expressed and secreted to the solution medium efficiently. Also the speciality of screted protein was conformed by Western-blot analysis with rat anti-CGH serum.CGH gene was inserted into pPIC9K plasmid and overexpression Pichia Pastoris yeast strains was constructed which realized efficient expression of CGH gene in yeast. Also the rat anti-CGH serum was obtained by immunizing rat with CGH Prokaryotic expressing product, and speciality of screted protein was conformed by the serum. This study sets a foundation for further research and application of recornbinant GH in Grass carp.