Prokaryotic Expression, Purification Of The Recombinant PGH And Its Bioactivity Study

Porcine growth hormone (pGH) gene had been correctly inserted into pGEX1-λT vector in order to construct the expression plasmid pGEX1-λT-pGH. The recombinant plasmid was also transformed into E.coli TOP 10F' competent cells. The positive clones including the expression plasmid were screened by colony PCR and plasmid PCR. We identified the recombinant plasmid by BamHI and EcoRI digestion. Fusion protein expression was induced by IPTG and expression situation was assayed by SDS-PAGE. A new protein band was found in SDS-PAGE with molecular weight of about 50 KDa which is consisted of a 24.4 KDa protein deduced from the pGH gene sequence and GST (26 KDa).The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit. The titer of rabbit anti-pGH serum from immunized rabbit was 1 : 6,400 determined through indirect ELISA by the rabbit anti-pGH serum as antibody and recovered pGH as antigen. The polyclonal antibody was purified by carylic acid and ammonium sulfate method. The specializations of antibody were identified by western blot and immunohistochemistry experiment. Results in this study showed that the purified antibody has only one band in SDS-PAGE gel and the concentration of it is 19.2mg/ml (tesed by Ultraviolet spectrophotometer) . Purified antibody can be specificallyimmunoactivated with the recombinant pGH (50KDa) and natural pGH (24 KDa) extracted from the pig pituitary. Positiveimmune signal of the prepituitary gland cell can be observed by purified antibody at the titer of 1:150.Research on denaturing and refolding of the inclusion bodies from expressed recombinant pGH expression strain in E.coli was carried out in order to test the bioactivity of purifed pGH protein. Complete lysation of harvested cell suspended in lysate buffer(Tris-HCl 50mmol/L, EDTA 0.5mmol/L, NaCl 50mmol; PH7.9) results from ultrasonic wave treatment. 20% sodium deoxycholate (DOC) solution was added to the buffer to reach 2% concentration. Centrifugateion was carried out at low temperature to get the settling. Lysate buffer with urea/guanidinium chloride (2mol/L) was used to resuspension and washing the sediment for two times. Denaturation result of inclusion bodies by 8mol/L urea solution was compared with that by 6mol/L guanidinium chloride solution at pH 8.0. The recovery rate of purifed pGH in Tris buffer at pH8.5 added with 0.5mol/L L-Arg achieved 37. 79%. The contration of recovery pGH is 194.5 μg/ml. ELISA receptor assay (ELISA-RA) result indicated that recombination pGH could be specifically immunoactivated with the receptor protein from pig hepatic membrane. It was verified that purifed pGH shoed a significant growth promoting effect on mice in one week after the only one injection.Above all, pGH gene had been successfully subcloned into expression vector pGEXl- A- T to construct the expression plasmid for pGH and its overexpression E.coli strains were screened. The rabbit anti-pGH serum for recombinant pGH was prepared by immunised rabbits with the purified recombinant pGH form the prokaryotic expression products. The speciality of purified anti-rabbit pGH antiserum was confirmed. The recombinant pGH was purified by denaturation and refolding technique and it showed a significant growth promoting effect on mice. This study founded bases for further research and application of recombinant GH in pig.