Prokaryotic Expression And Biological Activity Detection Of Chicken Interferon ?

Interferon(IFN)is a kind of cytokines produced by host cells,with function of broadspectrum antiviral,anti-tumor and improvement of the immune system under the effect of certain inducers.Its essence is secreting multifunctional glycoprotein.It was first discovered by Scientist Isaacs in the UK in 1957 who was doing research on the interaction between viruses.Its nature is protein.Induced by viruses,it can interfere with the viral replication,limit virus infection.According to the difference of sources of interferon,amino acid sequence and biological activities,interferon can be divided into three categories,namely?,?,? interferon.Type ? interferon mainly contains the lambda interferon.Study of human interferon lambda has been increasing,but there are few research Chicken interferon lambda(Ch IFN-?).So the mechanism of Ch IFN-? working with other interferon antivirals is not clear.Therefore,this study aimed at representing Ch IFN-? using the method of prokaryotic expression,exploring its biological activity,and working on both Ch IFN-? and Chicken interferon alpha(Ch IFN-?)with the method of fusion expression,exploring into the combination of biological activities of Ch IFN-? and Ch IFN?(Ch IFN-?+?).Research is represented as follows:Ch IFN-? and Ch IFN-?+? gene cloning and prokaryotic expression.According to the gene sequence from Gen Bank,2 pairs of primers were designed to amplify Ch IFN-? and Ch IFN-?.In so doing,the size of Ch IFN-? gene removed of signal peptide was expanded to about 492 bp and the size of Ch IFN-? gene removed of signal peptide,about 489 bp.Using overlapping extension PCR method,this research combined both Ch IFN-? gene and Ch IFN-? gene,the size of Ch IFN-?+? gene was expanded to about 1011 bp.Finally,with the p ET-32 a,prokaryotic expression vector of Ch IFN-? and Ch IFN-?+? was built.Massive expression and purification of protein Ch IFN-? and Ch IFN-?+?.The recombinant plasmid of prokaryotic expression was processed through inducing expression in BL21 competent cells.The results showed that protein both exists in the form of insoluble inclusion.Recombinant interferon of high biological activity were obtained by ultrasonic fragmentation,denaturation and renaturation and Ni column purification.According to the test of Western-bolt,the size of Ch IFN–? protein is about 37.2 k D,size of Ch IFN-?+? protein,about 56.9 k D.Antiviral activity research in vivo and in vitro.In vitro experiments,Ch IFN-? and Ch IFN-?+? well restrained vesicular stomatitis virus(VSV)and avian influenza virus(AIV)in chicken embryo fibroblast DF-1.In addition,Ch IFN-?+? also worked out for Newcastle diseases virus(NDV).Antiviral activities for VSV,NDV and AIV with Ch IFN-? in DF-1 cell are: 5.3 * 104 IU/mg,0,and 7.8 * 103 IU/mg;Antiviral activities for VSV,NDV and AIV with Ch IFN-?+? in DF-1 cell are: 3.2 * 104 IU/mg,4.6 * 104 IU/mg,1.5 * 105 IU/mg.Among them,antiviral activity of the fusion protein is higher than that of single Ch IFN-?.In vivo experiments,Ch IFN-? and Ch IFN-?+? had a certain degree of protection,which could effectively delay and reduce the discharge of virus.Compared with challenged group,the Ch IFN-? is low as for the quantity of discharging virus,and delayed 4 to 5 days of discharging virus,the rates of protection could reach 33%;Ch IFN-?+?,compared with challenged group,is also low as for the quantity of discharging virus,and delayed 3 ~ 5 days of discharging virus,the rates of protection can reach 25%.Antiviral effect research on chicken embryo.In chicken embryos experiment,Ch IFN-? and Ch IFN-?+? could provide protection,inhibit the virus propagation.Immune adjuvant effect research.Ch IFN-? and Ch IFN-?+? was used as immune adjuvant in vaccinate with Newcastle disease La Sota vaccine experiment.Compared with the control group,Ch IFN-? and Ch IFN-?+? could improve antibody titer rapidly in term of validity.The antibody titer can attain more than 1: 211.Vaccine joint immune adjuvant had effective antiviral activity.Through the amplification Ch IFN-? and Ch IFN-?+? gene and its prokaryotic expression,the purification of recombinant interferon was studied by means of biological activities.Study found Ch IFN-? and Ch IFN-?+? have good antiviral activity,as vaccine adjuvants have good immune enhancement effect,providing theoretical basis for clinical study interferon biological activities.