| Porcine Interferon(PoIFN)is a kind of broad-spectrum antiviral cytokine,which is an important antiviral factor in the body.It has shown excellent application potential as antiviral agents,and has attracted much attention in clinical studies of porcine diseases.Porcine Epidemic Diarrhea Virus(PEDV)is a virus that mainly causes acute diarrhea in pigs and has a high fatality rate especially for newborn and weaned piglets.Although the vaccine has been widely used,the protective effect is still not ideal.Studies have shown that interferon combined with vaccines and drugs can effectively prevent and treat PEDV.PoIFN type Ⅰ interferon has the best antiviral activity,but it is divided into many subclasses,among which PoIFN-α contains at least 17 subtypes,and the effects of each subtype in the anti-PEDV process are also different.It is necessary to explore the performance of each subtype in the anti-PEDV process for the development of novel anti-PEDV interferon preparations.In this study,the influence of codon optimization,medium,signal peptides and different prokaryotic expression vectors on the expression of soluble interferon was analyzed to determine the favorable factors to improve the expression of soluble interferon and establish a prokaryotic expression system for soluble interferon efficiently.At the same time,the bioactivities of IFN-α1～17 and IFN-β expressed by the system were identified,and the subtypes with excellent anti-PEDV activity were screened.The main contents are as follows:1.Construction,expression and purification of recombinant porcine interferonIn order to improve the prokaryotic expression of soluble type Ⅰ rPoIFN production,the IFN-α gene of ginger Quhai pig was cloned into prokaryotic expression vector pET30a(+)and expressed in E.coli.During the expression process,the factors affecting the prokaryotic expression of soluble interferon were analyzed through codon optimization,type of medium,signal peptide modification,different types of prokaryotic expression vectors and protein purification methods,and the protein expression level was analyzed by Western Blot test combined with the quantitative detection of ImageJ software and BCA protein.The results show that the gray value of WB strip after the warp code optimization is about 2.46 times of that before the codon optimization.The gray scale of WB bands in the HB-PET self-induction medium group was 1.3 times that in the 2×YT medium group and 3.82 times that in the LB medium group.The modification of signal peptide structure did not improve the expression of PoIFN-α.The concentration of IFN-α protein purified by nickel column is 750ng/μL,while the concentration of rPoIFN-α protein purified by magnetic beads can reach 1100ng/μL.The amount of soluble IFN-α expressed by PET30A-ELp and pET30a expression vectors was 705ng/μL and 734ng/μL,respectively,with no significant difference.The concentration of soluble IFN-α expressed by pET30a expression vector was 185ng/μL,and the difference was very significant.These results indicate that codon optimization,HB-PET self-induction medium and magnetic bead purification can improve the yield of soluble rPoIFN-αexpressed by pET30a vector.Different prokaryotic expression vectors expressed soluble rPoIFN-α at different levels.pET30a and the recombinant Pet30A-Elp vector inserted with lysotropic protein ELP expressed excellent vectors of soluble rPoIFN-α.The expression level of soluble rPoIFN-α was significantly higher than that of cold shock expression vector pCold-Ⅱ and pET22b vector containing secreted signal peptide pelB.2.Detection of antiviral activity of recombinant porcine interferonIn order to screen out the rPoIFN subtype with the best antiviral activity,17 subtypes of PoIFN-α and PoIFN-β of type Ⅰ were expressed and purified,and a higher concentration of recombinant porcine interferon was obtained.The toxicity of different subtypes of interferon on Vero cells was analyzed by CCK-8 method.The results showed that all rPoIFN subtypes had little effect on Vero cell proliferation at 100ng/μL concentration.By comparing the anti-VSV-GFP activity on Vero cells with human interferon standard,the interferon specific activity was measured.The results showed that rPoIFN-α2,8 had the highest specific activity,about 1～2×105IU/mg.The ratios of rPoIFN-αl,3,7,12,13,17 were 2～9.5×104IU/mg,and the ratios of rPoIFN-α6,10,11,14,15,16 were lower than those of rPoIFN-α5,9 and rPoIFN-β,while those of rPoIFN-α6,10,11,15,16 were 1～4×104IU/mg.Less than 2×103IU/mg,rPoIFN-α4 did not show antiviral activity against VSV-GFP in the concentration range of 100ng/μL.Finally,the antiviral activities of each subtype in PEDV prevention and treatment trials were determined.The results showed that rPoIFN-α6,17 had strong antiviral activity against PEDV compared with other subtypes in the prevention trial,and about 15-20 IU could effectively inhibit PEDV proliferation.rPoIFN-α3,7,8,11,12 require 20-50IU;rPoIFN-αl,2,9,10 require 50～100IU;rPoIFN-α13,14,15,16 showed weak anti-PEDV activity,requiring more than 100IU to effectively inhibit PEDV.rPoIFN-α4,5 and rPoIFN-β showed no significant anti-PEDV effect,and showed no antiviral activity at 100ng/μL concentration.In the treatment trial,the subtypes of interferon showed similar antiviral activity pattern,but the effective antiviral dose in the treatment trial was significantly higher than that in the prevention trial.These results indicate that rPoIFN-α1-17 and rPoIFN-β have significant differences in anti-PEDV activities,and rPoIFN-α6 and 17 have the highest antiviral activity against PEDV,showing potential for PED prevention and treatment.In summary,this study improved the prokaryotic expression levels of soluble rPoIFN-α1-17 and rPoIFN-β,identified their anti-PEDV activities,and screened out porcine interferon type Ⅰ subtypes with high anti-PEDV activity.These results provide data reference for the subsequent development of novel effective anti-PEDV interferon preparations. |
