| In the present study, two trials were conducted to evaluate the effects of immunization of a fusion protein including the second extracellular loop of sheepβ2-adrenoceptor (β2-AR) on the growth, slaughter performance and internal organs weight. The main studies as follows:The expression, purification and identify of the recombinant sheepβ2-AR: The recombinant plasmid pET32C-AR/Se harboring the second extracellular loop of sheepβ2-adrenoceptor gene were assigned to evaluate the effects of four factors including induction time, temperature, IPTG final concentration and rotation speed of shaker on expression level in E.coli BL21(DE3). The target protein was identified by SDS-PAGE and western blotting, and then purified by Ni-IDA column chromatography and gradient dialysis in guanidine hydrochloride at 4℃. The results showed that along with prolonging of IPTG induction time, the relative expression level of the recombinant protein increased gradually and achieved the peak 2 hs later, counting for 30.1% of the total bacterial protein. The expression level of the recombinant protein was between 27~30% of total bacterial protein when the final concentration of IPTG ranged from 0.01 to 2 mmol/L. The crest value of expression level was 30.5% of total bacterial protein when the final concentration of IPTG was 0.01 mmol/L. In four culture temperatures, the expression level of the protein reached the peak, 35.5% of the total bacterial protein at 25℃. When the rotation speed of shaker ranged from 100 to 200 rpm, the expression level of the protein fluctuated between 33~40%. The highest level of the foreign protein was achieved at 200 rpm, counting for 40% of the total protein. The SDS-PAGE and western blotting revealed that the fusion protein was in a form of inclusion bodies with the molecular weight approximately 26 KD. From the results, the conclusions could be drawn that the optimum culture conditions for expression of the second extracellular loop of sheepβ2-adrenoceptor in E.coli BL21(DE3) were when the recombinant bacteria grow to OD600 0.5 in LB fluid medium, IPTG added to a final concentration of 0.01 mmol/L, and induced at 25℃and 200 rpm for 120 min.Effects of active immunization of recombinant sheepβ2-AR on growth performance and serum biochemical indicator in Rats Forty five wistar female-rat, weighted about 175 g, were divided into three groups randomly, which including the control, immunity and agonist group. The control group immunized with adjuvant, and the immunity group immunized with the recombinant sheepβ2-adrenoceptor. The primary and booster immunization were carried out at the second and the fourth week from the initial respectively. At the third week, clenbuterol hydrochlorate was fed to the agonist group at the concentration of 3 mg/kg feed. Blood of all the rats were collected at the end of the sixth week to analysis serum parameters. Then all animals sacrificed to determine the slaughter performance and internal organs weight. The results showed that there was no significant difference between the control and immunity group. As compared with the control, active immunization againstβ2-AR increased the liver and lung weight by 13.83 % (P<0.05) and 18.46 % (P<0.05). The abdomen fat weight tended to decrease in immunity group, lowered by 12.20% (P=0.11). Serum urea nitrogen, triglyceride and cholesterol dropped by 9.68 % (P<0.05), 20.21 % (P<0.05) and 8.81 % (P=0.087) than the control group. In summary these data suggested that there were no significant effects of active immunization againstβ2-AR in the growth and intake, but it could reduce body protein catabolism and promote lipolysis in rats. |
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