| The baculovirus multi-gene expression system is the fourth largest expression system following expression systems such as bacteria,yeast and mammalian cells.It has more powerful function of post-processing modification than bacteria.Lower background interference than yeast;Compared with mammalian cells,it has lower cost,simple operation and low requirement on equipment.It has been improved from the initial plaque to Multi Bac,and the silkworm is easy to raise and the cost is low.Therefore,this study used a combination of Multi Bac and a silkworm bioreactor(Multi Bac-silkworm Bioreactor)to express recombinant proteins.Interferon(IFN)is a kind of glycoprotein with wildly biological activities including antiviral,anti-tumor and immunomodulatory.chIFN-?is a type III chicken interferon,whose functional receptor is mainly found in epithelial cells.Non-specific immunity is the first barrier to bacterial virus invasion,in which the skin and mucous membranes play an important role.It is especially important in anti-virus.Avian leukemia(AL)is a poultry infectious disease,and Avian leukosis virus(ALV)is one of the main causes of it.Sub group J(ALV-J),usually causes myeloid cytoma of the chicken,is highly pathogenic and infectious,which have seriously affected the development of white feather chicken breeding and even the whole poultry industry.At present,there are no effective vaccines and drugs to prevent and control them.(1)The chIFN-?gene,which the sequence of signal peptide was replaced with that of Bombyx mori nuclear polyhedrin GP64 and the DNA sequence of 6×His tag was fused to the terminal of it,was artificially synthesized.Subsequently,it was named Bm GP64sp-chIFN-?-His.(2)The eukaryotic transfer vector p FBDM-egfp-Bm GP64sp-chIFN-?-His was constructed using egfp as a fluorescent reporter gene.Recombinant Escherichia coli Bm Multi Bac/r Sw106-egfp-Bm GP64sp-chIFN-?-His was constructed by Tn7 transposons.Then,Bm N cells were infected with it.After fluorescence generation,blind passage for 3generations,Western-blot results showed that the P3 recombinant baculovirus BmNPV-chIFN-?with stable expression of recombinant chIFN-?was obtained.(3)The recombinant chIFN-?-His was successfully expressed by prokaryotic expression and detected by SDS-PAGE.The results showed that recombinant chIFN-?-His was present in the sample as inclusion bodies.A recombinant chIFN-?-His crude product was obtained by repeatedly washing the inclusion bodies,and the recombinant chIFN-?-His was emulsified with Freund's adjuvant,and the homemade mouse anti-chIFN-?polyclonal antibody serum was obtained by multiple immunization of the mice.(4)BmNPV-chIFN-?was injected into the 5th instar silkworm to express recombinant chIFN-?,which was subsequently purified by Ni-chelating affinity chromatography.Purified recombinant chIFN-?was identified by Western-blot using self-made mouse anti-chIFN-?polyclonal antibody serum.The results revealed that the recombinant chIFN-?was successfully purified.(5)The recombinant chIFN-?has antiviral activity on DF1 cells,and its antiviral activity is 3.45×10~2 UI/m L.(6)q PCR and ELISA were used to detect the biological function of recombinant chIFN-?.The results showed that recombinant chIFN-?has anti-ALV-J function in DF-1and LMH cells.After treatment with recombinant chIFN-?,the transcription levels of MX gene,Viperin gene and IFITM3 gene(ISGs gene family)were up-regulated in different degrees,It is speculated that recombinant chIFN-?may play an antiviral function by inducing the expression of MX gene,Viperin gene and IFITM3 gene in the organism.In conclusion,this experiment successfully expressed and purified the biologically active recombinant chIFN-?using Multi Bac-silkworm bioreactor,and it has significant anti-ALV-J function;Based on the characteristics of easy breeding,low cost and short cycle,this method can be used for preparation and mass production of recombinant chIFN-?,which has extensively prospects of research and application. |
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