Studies On Prokaryotic Expression Of Recombint Bifn-tau In E.coli And Its Application In Piglets

Interferons(IFN)are a type of glycoprotein with high biological activity,which produced and released by animal cells,they have a series of biological functions,especially in anti-virus,anti-cell proliferation and differentiation,immunoregulation,and so on.IFN-? is a unique interferon,belongs to type ? interferons,IFN-? as a embryo-derived pregnancy signal,and induced without virus.In addition to play an important biological function on during the establishment of outer,IFN-? also has a high activity of anti-virus,anti-tumor and immunoregulation,and so on.Compared to other type ? interferons,IFN-?has a lower cyotoxic with higher concentration,but it has similar activity of anti-virus.Currently,bIFN-? was almost in the form of inclusion body by the E.coli expression system in laboratory,and a small amount of bIFN-? with activity obtained only by the complex denaturation renaturation.Therefore,in this study,the rBoIFN-? gene was optimized according to E.coli codon bias,and tried obtain soluble by molecular chaperones and chemical chaperone co-expression,to lay the foundation for large-scale production and practical applications.Experiment 1.The prokaryotic expression of rBoIFN-? in E.coliThe rBoIFN-? gene was optimized according to E.coli codon bias,and then cloned into the cold expression vector pCold ?,then builded the rBoIFN-? expression vectors and expressed in E.coli.In order to obtain more rBoIFN-? in efficient expression in E.coli by optimizing the induction conditions.The results showed that rBoIFN-? gene was cloned into the expression vector cold pCold ? successfully,the recombinant plasmid called pCold?-rBoIFN-?,and then the recombinant plasmid was transformed into the E.coli expression system successfully.In the end,the target protein was obtained by inducing,and it's molecular weight was about 20 kD,but the target protein was still expressed as inclusion bodies.The optimal induction conditions of the expression of rBoIFN-? was IPTG concentration of 0.08 mmol/L,inducing temperature of 15 ?,inducting time of 12 h and inducting speed of 200 rpm.The results showed that,the bIFN-? was inclusion bodies protein which was expressed in E.coli expression system.Experiment 2.Effects of molecular chaperone on the expression formation of recombinant bIFN-? in E.ColiIn the experiment 1,rBoIFN-? gene was optimized and constructed an expression vector,but the target protein that was heterologous expressed in E.coli was inclusion bodies still.In the group 1,the rBoIFN-? expression vectors was transformed into E.coli BL21(DE3)with the molecular chaperone plasmids(pKJE7?pGro7?pGTf2 and pTf16).In the group 2,Different concentrations of chemical chaperones(Glycerin,Trehalose,D-mannitol,Ethanol,DMSO,Imidazole,L-arginine)co-expressed with positive recombinant E.coli which only had vector pCold ?-rBo1FN-?.The results showed that,the group 1,recombinant plasmid chaperone co-expression system has been successfully constructed by the double digestion method,two protein bands appeared in 50 kD and 20 kD place after the expression of the induction,the two protein bands molecular chaperones and rBoIFN-? protein respectively,and the target protein was inclusion bodies.The the group 2,seven kinds of chemical chaperones co-expressed with BL21 pCold ?-rBoIFN-? at the same conditions,and obtained a molecular weight of 20 kD inclusion body protein.The results showed that,molecular chaperones and seven kinds of chemical chaperone have no significant effect on soluble expression of rBoIFN-?.Experiment 3.The purification and antiviral effect of rBoIFN-?Inclusion bodies were collected in the experiment.Then these inclusion bodies were conducted by a serious operations like washing,dissolving and degeneration,purification and renaturation.The antiviral effect of the supermatant of treated inclusion bodies was tested by using MDBK-VSV micro cytopathogenic effect inhibition assay.The results indicated that the concentration of rBoIFN-? after purification was 2.7 times than that of rBoIFN-? before purification,and its purity was 97.2%.The recombinant soluble protein after renaturation got a concentration of 376 mg/L at the best-fit conditions and its activity Was 8.01×106U/mg.In conclusion,rBoIFN-? after a serious treatment above had a better antiviral effect.Experiment 4.The effects of rBoIFN-? on growth performance and morbidity rate of pigletsThere were three groups in the experiment.Pregnant sows in experiment group ? was treated with intramuscular injection of rBoIFN-? 2 mL,and the newborn piglets were intramuscularly injected of 0.5 ml and lmL rBoIFN-? at the seventh day after bom and the day before ablactation.The experiment group ? and the control group were treated with interferon SNTA and aseptic 0.9%NaCl solution respectively.The weights at bom and ablactation and morbidity rates were recorded detailedly.The results displayed that average daily gains(ADG)of three groups were respectively 0.226,0.221,0.207 kg/d.The rBoIFN-? group had a significant(P<0.05)ncrease in ADG compared with that of the control group.The morbidity rates of three groups were respectively 15.7,17.5,21.7%,and the rBoIFN-? group had a significant decrease compared with the control.So,rBoIFN-? had a positive effect in growth performance and reduced the incidence of piglets.