| A new type of goose parvovirus（Novel goose parvovirus,NGPV）,cause short beak-dwarfism syndrome（SBDS）,is one of the infectious diseases of waterfowl which has seriously affected the duck industry in China in recent years.The mortality rate of the disease is low,which is mainly characterized by dysplasia,short beak,bending of leg bone,shedding of feathers and so on,resulting in a high knockout rate and seriously affecting the income of breeding.The pathogen of the disease is difficult to be cultured by traditional methods,and the development of related vaccines is hindered.At present,Gosling plague（GP）vaccine and antibodies can only be used for prevention and control,but the effect is not stable.In recent years,it has been widely reported that lactic acid bacteria can induce the immune response of mucosal immune system and make up for the deficiency of humoral and cellular immunity.at the same time,lactic acid bacteria have the characteristics of probiotics and immune adjuvant function,so it is an ideal antigen delivery carrier.In this study,food-grade Lactococcus lactis NZ3900 and its matching expression plasmid pNZ8149 were used to construct recombinant lactic acid bacteria.VP2 protein,the main capsid protein of NGPV,was displayed on the surface of lactic acid bacteria with Pgs A protein,in order to develop a new preparation which can prevent and control NGPV.It is mainly divided into the following parts:1.Prokaryotic expression of VP2 protein:extract pathogen DNA and amplify VP2 gene.The VP2 gene was ligated into p ET32a plasmid and transferred into BL21 Escherichia coli.The strain was named p ET32a-VP2/BL21.The recombinant BL21 strain was induced to express protein by IPTG and the induction conditions were screened.The VP2 protein expressed by p ET32a-VP2/BL21 strain had His tag and was detected by Western blotting（WB）.The results showed that there were obvious protein bands at the expected 85k Da site,which proved that the VP2 protein was successfully expressed.SDS-PAGE assay confirmed that VP2protein was mainly expressed in the form of inclusion bodies.The purified protein was verified by SDS-PAGE,which showed that the heterozygosity was significantly reduced and obvious protein bands appeared at the expected 85k Da position.2.To establish a new indirect ELISA method of goose parvovirus VP2 protein for subsequent detection of related VP2 antibodies.Firstly,the conditions of ELISA reaction were optimized.42 NGPV negative duck serum samples were detected according to the optimum reaction conditions,and the critical value of negative and positive was determined to be 0.106.The results of repetitive test,sensitivity test,specificity test and clinical sample test show that each index of this method is good and can be used for practical detection.3.Preparation of polyclonal antibody:Kunming mice aged 6-8 weeks were immunized with VP2 protein mixed with Freund’s complete adjuvant/Freund’s incomplete adjuvant.At the end of immunization,eyeballs were extracted and blood was taken to separate serum,and the titer of polyclonal antibody was detected by ELISA.The results showed that the titer of polyclonal antibody was more than 100000 times,which could be used in related protein immunity experiments.4.Construction and induced expression of recombinant Lactococcus lactis surface display system pNZ8149-Pgs A’-VP2/NZ3900:linearized pNZ8149 plasmid,Pgs A’gene and VP2 gene were connected to construct pNZ8149-Pgs A’-VP2 recombinant plasmid and transferred into NZ3900 Lactococcus lactis by electroporation.The strains with correct recombinant plasmid were selected by Elliker medium and sequenced.The strain with correct recombinant plasmid was named pNZ8149-Pgs A’-VP2/NZ3900.After the recombinant lactic acid bacteria were induced by nisin（nisin,Nisin）,the expression of VP2 protein was detected by WB.The results showed that pNZ8149-Pgs A’-VP2/NZ3900 recombinant lactic acid bacteria successfully expressed VP2 protein on the cell surface.5.Study on the immune characteristics of recombinant Lactococcus lactis pNZ8149-Pgs A’-VP2/NZ3900:in order to avoid the interference of NGPV,GPV and other viruses in the environment,6-week-old chickens which are not susceptible to the virus were selected as immune experimental animals.The experimental group was fed with pNZ8149-Pgs A’-VP2/NZ3900 recombinant bacteria solution,and the control group was fed with NZ3900solution containing pNZ8149 plasmid.The immune cycle was as follows:continuous administration for 5 days,interval of one week,then continuous administration for 5 days,and the end of the experiment on the 21st day.Serum,small intestinal mucosa,esophageal mucosa and tracheal mucosa were collected to detect VP2 antibody,total Ig A,IL-2 and IFN-γantibody.The results showed that a high level of VP2 antibody was detected in both esophageal mucosa and intestinal mucosa,indicating that the levels of cytokines in serum and mucosa and the total Ig A level in small intestinal mucosa increased significantly,indicating that the VP2 protein displayed on the surface of recombinant lactic acid bacteria successfully played the role of immune antigen.6.Study on the effect of recombinant Lactococcus lactis pNZ8149-Pgs A’-VP2/NZ3900on intestinal microflora of Cherry Valley ducklings:1-day-old Cherry Valley ducklings were selected and the control group was not intervened.The experimental group was continuously fed with pNZ8149-Pgs A’-VP2/NZ3900 recombinant bacteria solution,1×1010CFU,once a day,and stopped after a week.On the 14th day,the contents of cecum were dissected and the changes of lactic acid bacteria,bifidobacteria and intestinal pathogenic bacteria were detected.The results showed that there was no significant change in the number of lactic acid bacteria,the number of bifidobacteria increased significantly,and the number of Escherichia coli in the experimental group was significantly lower than that in the control group.it is proved that the recombinant Lactococcus lactis constructed in this study is effective in maintaining the balance of intestinal flora and counteracting pathogenic bacteria.7、One-day-old Cherry Valley ducklings with good health were randomly divided into 4groups.The normal group was not immunized and raised in isolation,while the control group was only treated with challenge.The pNZ8149/NZ3900 group was treated with pNZ8149/NZ3900 Lactococcus lactis,and the pNZ8149-Pgs A’-VP2/NZ3900 group was treated with pNZ8149-Pgs A’-VP2/NZ3900 recombinant Lactococcus lactis.The oral dose of the strains was 1×1010CFU,once a day.The mice were immunized at 1 day old,continuously for 7 days,and challenged respectively at 4 days old.The body weight changes of ducklings at1,5,9 and 14 days old and the death and pathological changes of each group at 14 days old were recorded respectively to judge the immune protection effect of recombinant lactic acid bacteria.The results showed that the recombinant Lactococcus lactis expressing new duck parvovirus VP2 protein had good immune protection,which could effectively prevent the emergence of molting and dysplasia caused by new goose parvovirus and reduce mortality.To sum up,the recombinant lactic acid bacteria constructed in this study can play the role of immune antigen and has the function of immune protection,and the recombinant lactic acid bacteria are effective in maintaining the balance of intestinal flora and counteracting pathogenic bacteria,indicating that the recombinant lactic acid bacteria have been successfully constructed and are expected to become a new preparation for the prevention and control of NGPV. |
