Cholera toxin B subunit(CTB)can enhance antigen presentation and promote T cell pro-liferation,B cell differentiation and B cell isotype conversion.Moreover,woodchuck hepatitis virus post transcriptional regulatory element(WPRE)can enhance gene expression efficiency by optimi-zing RNA polyadenylation,denuclearization and/or translation.In order to construct porcine epi-demic diarrhea virus like particles(VLPs)chimerized with CTB and WPRE and evaluate their im-munogenicity,the G Ⅱ type PEDV S gene,combined with the elements promoting the protein ex-pression and enhancing immune effects,was synthesized by the company and cloned into pET32a(+).Af-ter double enzyme digestion and gel recovery,the gene named as TSCW was cloned into pFastBacl to construct the recombinant plasmid pFastBac-TSCW.pFastBac-TSCW was further transformed into DH10Bac competent cells to obtain recombinant bacmid Bacmid-TSCW.Subsequently,the Bacmid-TSCW was transfected into sf9 cells to obtain recombinant baculovirus BV-TSCW.After co-infection of BV-TSCW and BV-M into sf9 cells,viral like particles VLP-TSCW was obtained and used to immunize mice to evaluate its immunogenicity.The results showed that the recombi-nant plasmid pFastBac-TSCW and bacmid Bacmid-TSCW were successfully constructed.After transfection of sf9 cells with recombinant baculovirus,significant cytopathic effects were observed.PCR and Western blot results showed that the recombinant baculoviruses existed stably in sf9 cells and the target proteins was also expressed stably.In addition,the electron microscopy results showed that BV-TSCW and BV-M successfully assembled into viral like particles VLP-TSCW.Furthermore,ELISA results indicated that VLP-TSCW induced high level specific antibodies.The above results laid the foundation for further optimization,design and development of PEDV VLPs subunit vaccines.

Cholera toxin B subunit(CTB)can enhance antigen presentation and promote T cell pro-liferation,B cell differentiation and B cell isotype conversion.Moreover,woodchuck hepatitis virus post transcriptional regulatory element(WPRE)can enhance gene expression efficiency by optimi-zing RNA polyadenylation,denuclearization and/or translation.In order to construct porcine epi-demic diarrhea virus like particles(VLPs)chimerized with CTB and WPRE and evaluate their im-munogenicity,the G Ⅱ type PEDV S gene,combined with the elements promoting the protein ex-pression and enhancing immune effects,was synthesized by the company and cloned into pET32a(+).Af-ter double enzyme digestion and gel recovery,the gene named as TSCW was cloned into pFastBacl to construct the recombinant plasmid pFastBac-TSCW.pFastBac-TSCW was further transformed into DH10Bac competent cells to obtain recombinant bacmid Bacmid-TSCW.Subsequently,the Bacmid-TSCW was transfected into sf9 cells to obtain recombinant baculovirus BV-TSCW.After co-infection of BV-TSCW and BV-M into sf9 cells,viral like particles VLP-TSCW was obtained and used to immunize mice to evaluate its immunogenicity.The results showed that the recombi-nant plasmid pFastBac-TSCW and bacmid Bacmid-TSCW were successfully constructed.After transfection of sf9 cells with recombinant baculovirus,significant cytopathic effects were observed.PCR and Western blot results showed that the recombinant baculoviruses existed stably in sf9 cells and the target proteins was also expressed stably.In addition,the electron microscopy results showed that BV-TSCW and BV-M successfully assembled into viral like particles VLP-TSCW.Furthermore,ELISA results indicated that VLP-TSCW induced high level specific antibodies.The above results laid the foundation for further optimization,design and development of PEDV VLPs subunit vaccines.

Objective To investigate the clinical application and efficacy of DNA immune absorption in patients with lupus interstitial pneumonia.Methods to collect randomized 18 patients with lupus patients with pneumonia were enrolled in the study and randomly divided into immunoadsorption group and traditional CTX treatment group,in order to observe the ESR,CRP,ANA quantitative monitoring at different time,pulmonary function test (diffusing capacity of the lung for carbon monoxide,DLCO),6 min walking distance,procalcitonin (PCT).The difference between groups was statistically analyzed and the effect of DNA immunization was discussed.Results There were significant differences between immunoadsorption group and control group in ESR at the different time points before and after the treatment (Fgroup =7.841,P＜0.05;Fcross =6.512,P ＜0.05;Finteraction =10.421,P＜0.05),CRP(Fgroup =6.995,P＜0.05;Fcross=5.847,P＜0.05;Finteraction =8.847,P＜ 0.05) and ANA quantitative monitoring (FgrouP =12.336,P ＜ 0.05;Fcross =11.214,P ＜ 0.05;Finteraction =15.847,P＜0.05).At 1 and 2 weeks after treatment,CRP and ESR of the immunoadsorption group began to decrease,and the difference was statistically significant compared with those before treatment (P ＜0.05),while the difference between the control group and the treatment group was statistically significant after 4 weeks (P＜0.05).After 2 weeks of treatment,there was a significant difference in ANA quantitative monitoring between the immunoadsorption group,compared with that before treatment.There was a significant difference between the control group before treatment and the 6 months after treatment (P＜0.05).There was a significant difference between the immunoadsorption group and the control group in pulmonary function test (FgrouP =6.222,P＜ 0.05:Fcross =7.154,P＜ 0.05:Finteraction =8.527,P ＜ 0.05),6 min walking distance (FgrouP =8.669,P＜ 0.05;Fcross =7.154,P ＜ 0.05;Finteraction =11.547,P＜ 0.05) and PCT (FgrouP =5.621,P ＜0.05;Fcross =4.125,P ＜ 0.05;Finteraction =7.554,P ＜ 0.05.The pulmonary function and 6 min walking distance of 2-week treatment in the immunoadsorption group.There showed a significant difference compared with that before treatment.The difference between the control group after 4 weeks of treatment and that before treatment was statistically significant (P=＜0.05).There was a significant difference between the 2 weeks PCT treatment in the immunoadsorption group and that before treatment (P＜0.05).There was a significant difference between the control group after 3 months of treatment and before treatment (P ＜ 0.05).Conclusion The treatment of lupus interstitial pneumonia in traditional regimens is ineffective,and the efficacy of DNA is better than that of conventional regimens,and reduces the risk of infection.