Coexpression Of Growth Hormone Releasing Factor And Pituitary Adenylate Cyclase Activating Polypeptide Genes

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)and (GrowthHormone Releasing Factor,GRF)are synthesized and secreted by hypothalamus,which can stimulate pituitary to synthesize and secrete growth hormone, thus,increasing GH concentration in animal and human bodies.PACAP coding gene was obtained with chemical synthesis and PCRamplification. The eukaryon expression vectors, pIRES1-PACAP, pIRES1-GRF andpIRES1-PACAP-GRF, were constructed respectively, then expressed in CHOsuccessfully cells introduced by Lipofectin,validated by RT-PCR and Western bloting.Biological activity of the expressed products was detected in rat in vivo. PLGAmicrospheres containing recombinant plasmids were prepared. After muscularinjection of PLGA encapsulated plasmid in mice and rabbits, it was found that thepromotion of growth by the double gene co-expression (pIRES1-PACAP-GRF) wassuperior to the single gene, rabbit IGF-1was also higher.1.PACAP gene was synthesized chemically and amplified by PCR, 205bp inlength,coding signal peptide and thirty-eight amino acids of PACAP , with addition ofcohensive ends of SmaⅠ,XbaⅠ.After amplification for five times by PCR, theproducts was purified in 2.5% agarose gel electrophoresis, then digested with SmaⅠ,XbaⅠ.Digested fragments were ligated to pIRES1-neo with the same digestion,andtransformed to competent JM109, positive recombinants were selected and identifiedby PCR, restriction enzyme digestion and sequencing. 2. GRF gene was amplified by PCR taking pcDNA3-GRF(1-32) plasmid astemplet.The products were purified in 2.5% agarose gel electrophoresis. Doubledigestion fragments of PCR products (EcoRⅠ,BamHⅠ)and pIRES1-neo wereprepared and ligated,then transformed to competent JM109, positive recombinantswere selected and identified by PCR, restriction enzyme digestion and sequencing. 3. pIRES1-neo –GRF plasmid and PACAP fregments digested with SmaⅠ,XbaⅠ were prepared and ligated , then transformed to competent JM109, positive recombinants were selected and identified by PCR, restriction enzyme digestion and sequencing . 4. The three expression vectors were transfected by Lipofectin to prepared CHOcells in 60% confluent according to provided protocols,then total RNA was extractedfrom transfected CHO cells,the positive result was obtained by RT-PCR, Dot-ELISAand Western blot. Rats were treated with 0.5ml of transfection supernatant by muscleinjection,it was found that there was no signicicant difference in serum IGF-1concentration among groups(P﹥0.05),but IGF-1concerntration in P-G-P group wasthe highest; After 8 hours, IGF-1concerntration in P-G-P group was significantlyhigher than the other groups(P﹤0.05),increasing by 37.52pg/ml,23.25 pg/ml and22.46 pg/ml than those groups injected with saline ,P-G microspheres ,P-Pmicrospheres, respectively. 5.Three expression plasmids were extracted and poly lactic-co-glycolic acid(PLGA) microspheres encapsulating plasmids were prepared by double emulsion-inliquid evaporation process. Entrapment efficiency was achieved to 99.79%, meanparticle size 2.41μm and the yield 73.12%; Mice were muscularly injected withplasmid microspheres(1mg plasmid/ kg of BW), bare plasmid(1mg / kg of BW) andsaline. After 20 days, the mean accumulative body weight gain in P-G-P microspheres...