. 2006, 24(0608): 36-39.
PACAP coding gene was obtained with chemical synthesis and PCR amplification. The eukaryon expression vectors, pIRES1-PACAP, pIRES1-GRF and pIRES1-PACAP-GRF, were constructed respectively, then the three expression vectors were transfected by Lipofectin to prepared CHO cells. The total RNA was extracted from transfected CHO cells,and the positive result was obtained by RT-PCR, Dot-ELISA and Western blot. Rats were treated with 0.5 ml of transfection supernatant by muscle injection. After 8 hours, IGF-1concerntration in P-G-P group was significantly higher than that of the other groups(P﹤0.05). Three expression plasmids were extracted and poly lactic-co-glycolic acid (PLGA) microspheres encapsulating plasmids were prepared by double emulsion-in hliquid evaporation process. Mice were muscularly injected with plasmid microspheres, bare plasmid and saline. After 20 days, the mean accumulative body weight gain in P-G-P microsperes group was the highest, increased by 64.71%(P<0.01), 28.78%(P<0.01), 32.72%(P<0.01), and 20.78%(P<0.05)to groups injected with saline, P-G microspheres, P-P microspheres and bare plasmids, respectively. Rabbits were treated with three plasmid microspheres(1 mg plasmid/kg of BW)and saline by muscular injection. After 30 days, the mean accumulative body weight gain in plasmid microspheres groups were significantly higher than that of the group injected with saline. Among groups, the accumulative body weight gain in P-G-P group was the highest,increased by 81%(P<0.01), 15%(P<0.05)and 7%(P>0.05) to the saline group, P-G microspheres group and P-P microspheres group, respectively. The concentrations of serum IGF-1 increased in all plasmid microspheres injection groups, which were significantly higher than that of the saline injection group. We draw a conclusion that GRF and PACAP co-expression in animal body do increase IGF-1 concentration and improve animal growth,whose biological activity might be more powerful than GRF and PACAP single gene.
