Analysis Of The Gene Of Pseudosciaena Crocea Growth Hormone And Its Expression In The Escherichia Coli

Problems such as low growth rate and low conversion rate of feedstuff exist inthe culturing of Pseudosciaena crocea and other economical marine fishes. Therefore,it is economically important to exploit some genetic products of fish itself to improvethe properties of feedstuff. Fish growth hormone (fGH) is a polypeptide produced bythe pituitary cells of teleosts. It has the capacity to enhance the appetite and feedconversion efficiency. However, the content of native fGH in pituitary is extremelylow and the expenditure of purification is too high. Fortunately, it is possible toproduce recombinant fGHs abundantly. And they have been proved in many cases thesame function as the native ones without any side effect to human. Therefore, it isvaluable to investigate the production of recombinant Pseudosciaena crocea GH(pcGH). In this research, the sequence of a newly cloned pcGH gene was analysed. Thenrecombinant pcGH was produced both in intracellular and periplasmic manner. Andthe factors effecting the expression level of the Escherichia coli were discussed. The newly cloned pcGH showed high homogeneity to pcGH and Sciaenopsocellata GH from GenBank in different regions respectively. Therefore this gene waspresumed to be the hybrid of several species of fish. This supposition will contributeto find out the conserved regions of fGH. It'll also help to investigate the influence ofgenetic mutation of GH on the growth of Pseudosciaena crocea. The pcGH gene was cloned into pET-22b(+) for intracellular expression.Recombinant plasmid (pG2) were transformed into E. coli BL21(DE3) andRosetta(DE3). SDS-PAGE analysis showed that a specific protein band with the samemolecular weight as theoretical one of pcGH was induced in pG2/Rosetta(DE3); butin the case of pG2/BL21(DE3), this band was difficult to be found. Therefore, it'sconcluded that Rosetta(DE3) can help to lighten the effect of rare codon on theexpression level in E. coli. The product of pG2/Rosetta(DE3) was proved soluble,then it was purified to be electrophoretically pure by ammonium sulfate precipitationand cation exchange chromatography. Another pair of primer was designed to insert the gene of pcGH into the IIdownstream of pel B signal peptide in pET-22b(+). The recombinant plasmid wastransformed into E. coli Rosetta(DE3). With IPTG induction, part of the recombinantpGH was secreted into the periplasm of transformants. Factors effecting the expression level of pcGH in E. coli, both on transcriptionlevel and translation level, were discussed integrallty and systemically. Approches toimprove the expression level were put forward . These works may have potential value in the production and application ofpcGH.