| Gosling plague is a kind of infectious disease with high morbidity and high mortality,which posted a serious threat to goose breeding industry in China.At present,there is no effective therapeutic drug for gosling plague,which mainly relies on Immunoglobulin of yolk and high serum-free serum,so the development of new drugs is of great significance.Interferon(IFN)is an important immune cytokine in vivo that interferes with virus reproduction.It is mainly induced by viruses,bacteriophages and synthetic nucleotides(such as poly-cells),with broad spectrum of antiviral activity.IFN inhibits a variety of viruses such as DNA viruses and RNA viruses,and also has anti-proliferative,anti-tumor and immunoregulatory functions.In this study,goose interferon alpha(goIFNα)was cloned into E.coli-Lactobacillus shuttle expression vector pSIP409-pgsA’,and electro transformed into Lactobacillus plantarum NC8 to obtain the new functional Lactobacillus with antiviral effect.This study is of great significance for the preparation of antiviral novel microecological for the prevention and treatment of gosling plague and for ensuring animal production safety and food safety.The specific research programme and results are as follows:First,we optimized the interferon gene published on GenBank according to the preference codon of Escherichia coli.The coding sequence of goIFN was synthesized by biological company,and the fragment of goIFN was linked to the prokaryotic expression vector pET28-a,and the prokaryotic expression plasmid pET28-a-goIFNα was successfully constructed.Then,the prokaryotic expression plasmid was transferred into Escherichia coli BL21(DE3)competent cells,and SDS-PAGE and western-blot detection were performed after IPTG induction.Additionally,the results of SDS-PAGE and Western-blot indicated that the molecular weight of the recombinant goIFN protein was about 18 kd,the same size as expected,and had good reactogenicity.The purpose strip was recovered and mixed with PBS,then injected into mice,and the serum was recovered 15 days later.The results of Western-blot indicated that anti-goIFN serum was successfully produced.According to the published goose interferons alpha sequence of geese on GenBank forward and reverse primers were designed upstream and downstream of the complete open reading frame,and Xba I and HindⅢrestriction sites were added to the forward and reverse primers,respectively.PCR amplification was performed using plasmid pMD18-T-goIFNα as template,and the goIFNα gene fragment was obtained and connected with the Escherichia coli-Lactobacillus shuttle expression vector pSIP409-pgsA’ to construct a shuttle expression plasmid pSIP409-pgsA’-goIFNα.The prokaryotic expression plasmid was electro transformed into Lactobacillus plantagensis NC8 to construct the the new functional Lactobacillus NC8-pSIP409-pgsA’-goIFNα(abbreviated as pp-goIFNα/NC8).The plasmids were extracted,verified by PCR and double enzyme digestion,and the plasmids were sequenced.The results showed that the new functional Lactobacillus was successfully constructed.The new functional Lactobacillus were induced by S.sakasin-P(SppIP)and then subjected to ultrasonic disruption and repeated freezing and thawing.Western-blot analysis showed that the new functional Lactobacillus had expressed goIFNα protein,and the immunofluorescence test also proved that the expression of goIFN on the bacterial surface had good reactogenicity.The antiviral effect of the new functional Lactobacillus was detected by goose embryo fibroblasts(GEF)in vitro.Firstly,the antiviral effects of the new functional Lactobacillus at different concentrations(10:1,80:1,160:1)were determined.The antiviral effect was the best when the concentration ratio between the new functional Lactobacillus and cells was 80:1.The cells were then incubated with the new functional Lactobacillus at a ratio of 80:1,and the groups were set as follows: DMEM blank control,GPV control,interferon protein control,NC8 vector group(NC8pSIP409-pgsA’,pp/NC8),pp-goIFNα/NC8+GPV group(ie,first added the new functional lactic acid bacteria NC8-pSIP409-pgsA’-goIFNα was added to gosling plague virus GPV),GPV+pp-goIFNα/NC8 group(ie,the new functional lactic acid bacteria pp-goIFNα/NC8 was added after adding goose plague virus GPV).The results showed that the pp-goIFNα/NC8+GPV group,the GPV+pp-goIFNα/NC8 group,and the recombinant protein group had good antiviral effects as compared with the empty vector group.In addition,we also determined the relative expression levels of the cellular antiviral factors goIFNα and goIFN-β as well as the interferon stimulating factors MX,OAS and PKR genes.The results showed that compared with the pp/NC8 group,the goIFNα+GPV group,GPV+pp-goIFNα/NC8 group and recombinant protein were able to up-regulate the expression levels of goIFNα and goIFN-β genes in GEF,thereby up-regulating the mRNA level of intracellular interferon-stimulated gene(ISG)and protecting the cells from being infected with GPV.The new functional Lactobacillus were orally administered for 3 days in the 3 day old goslings.On the 4th day,the goslings were challenged with GPV,and the body weight and survival rate was recorded.After immunization,tissue samples were taken after immunization and the relevant indexes were detected.The serum levels of antiviral factors IFNα and IFN-β were measured in peripheral blood,and the gene expression levels of the cell antiviral factors goIFNα and goIFN-β and the interferon stimulating factors MX,OAS and PKR genes in the spleen were measured.Pathological sections were taken from the liver,heart,spleen and intestine to observe histopathological changes.The results showed the survival rates of Normal group,GPV group,pp/NC8+GPV group,pp-goifn /NC8+GPV group,GPV+ pp-goifn /NC8 group and IgY group were 100%,30%,40%,50%,70% and 40%,respectively.After extracting viral DNA and total RNA from spleen,the results showed that compared with the control group,the new functional lactic acid bacteria pp-goIFNα/NC8 couldignificantly reduce the expression of GPV DNA gene and enhance IFNα and IFN-β in gosling spleen.The level of gene expression was thereby up-regulated by various antiviral cytokines such as MX,OAS,PKR and the like.Histopathological changes were most severe in GPV group,including liver and heart.There were severe histopathological changes in the spleen and small intestine,but no histopathological changes were observed in the pp-goifn /NC8+GPV group,GPV+ pp-goifn /NC8 group and IgY group.In summary,this experiment successfully prepared mouse anti-goIFNα serum,constructed the new functional lactic acid bacteria pp-goIFNα/NC8,and anchored goIFNα protein on the surface of lactic acid bacteria.The results showed that the new functional lactic acid bacteria can reduce histopathological damage and inflammation lesions,reduce the replication of the virus in the body,and increase the antiviral effect of the body,which is of great significance for the development of biological products for treating goose plague. |
PDF Full Text Request