| Porcine circoviru(PCV),a member of the genus Circovirus in the family of Circovirus,which is divided into two serotypes,PCV1 and PCV2.PCV1 is non-pathogenic,but PCV2 can cause post weaning multi-systemic wasting syndrome in weaning piglets 、 congenital tremor 、 porcine dermatitis and nephropathy syndrome and other diseases,the mortality rate of swine herds at acute outbreaks can reach 50%.PCV2 brings serious harm to the pig industry.There are about 11 open reading frames(ORFs)in the genome of PCV1 and PCV2.The copy protein Rep of PCV is encoded by ORF1,and the viral capsid protein Cap is mainly encoded by ORF2 gene.The structural protein Cap encoded by ORF2 is the main protective antigen of the virus,with the close relationship to the virus immune mechanism,and is the preferred protein for the clinical development of PCV subunit vaccines.Lactococcus lactis is a host vector for subunit vaccines because of the small amount of exogenous protein,and the recombinant bacteria has the advantage that it can be fed directly without purification.In this study,recombinant Lactococcus lactis expressing Cap protein was constructed by using p H-inducible expression vector p AMJ399 with the Lactococcus lactis as a host strain.This strain can also express Cap protein and playing the role of prebiotic effect of lactic acid bacteria at the same time,and as a recombinant subunit live vector vaccine against porcine circovirus disease,which provides a new idea for the study of PCV new oral vaccine.In this study,the PCV2 Cap gene fragment containing two restriction sites Bgl II and Pst I and removing the nuclear localization signal sequence was amplified by PCR of about 579 bp.The Cap gene was ligated with the plasmid p AMJ399 which was expressed by Lactococcus lactis and transforred into Escherichia coli competent cell TG1,the recombinant plasmid p AMJ399-Cap was obtained and it was transferred into Lactococcus lactis competent cell MG1363 to obtain recombinant Lactococcus lactis p AMJ399-Cap/MG1363.SDS-PAGE showed that the recombinant bacteria p AMJ399-Cap/MG1363 after induction appeared at about 28 k Da with the expected result of the band size.Western blot analysis of PCV2 monoclonal antibody 3C11 supernatant showed that the recombinant bacteria p AMJ399-Cap/MG1363 appeared a specific reaction band at about 28 k Da,indicating that Cap protein was expressed in recombinant Lactococcus lactis p AMJ399-Cap/MG1363.Indirect immunofluorescence assay and ELISA test showed that the Cap protein could be secreted into the culture medium and also distributed on the cell surface.The biosafety evaluation of the recombinant Lactococcus lactis p AMJ399-Cap/MG1363 was carried out.The survival performance of the recombinant bacteria in the simulated digestion environment and the retention in the digestive tract after oral administration showed that the survival rate of the recombinant bacteria was 63.09% at p H 3.5 in the gastric juice environment for 5 hours,and the survival rate of the recombinant bacteria can survive more than 24 hours,and had good tolerance to 5% salt concentration.At 4 hours after the single intragastric administration of the recombinant bacteria,the jejunum and colon were detected strong surviving signal in mice,the fluorescence intensity of the recombinant bacteria was significantly higher than that of the control group;the fluorescence signal of each intestine was weak at 8 h,indicating that the recombinant bacteria survived less;after 4 days of continuous intragastric administration,there was a significant fluorescence signal in the jejunum,ileum and colon on the 5th day,indicating that there were survivors of the recombinant bacteria;on the 6th day and 11 th day of the test,there was no fluorescence signal in the jejunum and ileum,however there was obvious fluorescence signal in the colon.It was found that the recombinant bacteria could remain in the intestine for more than 10 days.To further explore the immune effect of recombinant Lactococcus lactis,this study used mice as experimental animals for oral immunization.The experiment was divided into three groups: the experimental group was orally immunized with the recombinant bacteria p AMJ399-Cap/MG1363 with the dose of 4 × 107CFU/only;the control group was divided into empty vector group and PBS group,the mice were orally immunized with empty vector bacteria p AMJ399/MG1363 with the dose of 4 × 107CFU/only and the same volume of PBS respectively.Immunization program: immune twice,each time the immune interval of 2 weeks,each time continuous immunization 3 days,once a day immune.Feces、serum and intestinal mucus samples were collected at different times before and after immunization,the levels of specific s Ig A in feces、specific Ig G levels in serum and specific s Ig A levels in intestinal mucus were measured by ELISA method.The results showed that recombinant bacteria were orally immunized to mice,which could increase the daily feed intake and increase the daily gain;the s IgA and Ig G levels of mice immunized with recombinant bacteria p AMJ399-Cap/MG1363 were higher than those of the control group,indicating that oral Lactococcus lactis can not only induce local mucosal immune response,but also stimulate the body to produce systemic humoral immune response.The recombinant Lactococcus lactis p AMJ399-Cap/MG1363 which was constructed by this study could stably express PCV2 Cap protein and had good safety.Oral immunization can induce the mucosal and humoral immune response of the body,which has a good protective effect on the animal body.Through the development of this study,it has laid a foundation for the application of recombinant Lactococcus lactis in oral vaccine. |
PDF Full Text Request