| African swine fever(ASF)is an acute,severe and highly contact animal infectious disease caused by African swine fever virus(ASFV).Since ASF was first introduced into China in 2018,it has caused huge economic losses to China’s pig industry.Therefore,it is urgent to develop a safe and efficient ASFV vaccine.Because p30,p54,p72 structural proteins can induce immune response in vivo.Therefore,it can be used in the development of genetic engineering vaccine.In this study,Lactococcus lactis(L.Lactis)was used as antigen presenting vector to construct genetically engineered bacteria expressing ASFV p30,p54,p72 proteins and LTB subunit,the immunogenicity of orally immunized recombinant Lactococcus lactis in rabbits.was farther evaluated.The aim is to provide theoretical basis for the development of oral ASFV subunit vaccine.TestⅠ:According to the gene sequences of GenBank Nos.ASFV p30(M96354.1),p54(MZ812353.1)and p72(MN886930.1),p30-His,p54-His,p72-His,p30-LTB-His,p54-LTB-His and p72-LTB-His were separately cloned into expression vector pMG36e by truncating genes and synthesizing gene fragments carrying Sal I and Hind III restriction sites.Recombinant plasmids pMG36e-p30-His,pMG36e-p54-His,pMG36e-p72-His,pMG36e-p30-LTB-His,pMG36e-p54-LTB-His and pMG36e-p72-LTB-His were constructed.The recombinant plasmids were electrically transformed into L.lactis MG1363 competent cells,and the recombinant L.lactis were successfully constructed.They were named L.lactis MG1363/pMG36e-p30-His,MG1363/pMG36e-p54-His,MG1363/pMG36e-p72-Hisand MG1363/pMG36e-p30-LTB-His,MG1363/pMG36e-p54-LTB-His,MG1363/pMG36e-p72-LTB-His.SDS-PAGE and Western blot analysis showed that the target proteins p30,p54,p72 and fusion proteins p30-LTB,p54-LTB,p72-LTB could be expressed in recombinant L.lactis.TestⅡ:Forty female New Zealand white rabbits with similar body weight were randomly divided into4 groups with 10 rabbits per group.Group 1 was PBS-treated,group 2 was MG1363/pMG36e,group 3 was vaccine-treated without LTB(recombinant L.lactis MG1363/pMG36e-p30-His+MG1363/pMG36e-p54-His+MG1363/pMG36e-p72-His combined immunization),group 4 was the vaccine-treated group with LTB(recombinant L.lactis MG1363/pMG36e-p30-LTB-His+MG1363/pMG36e-p54-LTB-His+MG1363/pMG36e-p72-LTB-His combined immunization).Group 1,each of rabbits was given the 3 mL of PBS(pH=7.4),group 2,each of rabbits was given the same dose of concentration 1.0×10~8CFU/mL MG1363/pMG36e,group 3,each of rabbits was orally immunized with same dose and concentration of recombinant L.lactis MG1363/pMG36e-p30-His,MG1363/pMG36e-p54-His,MG1363/pMG36e-p72-His,1 mL for each,group 4,each of rabbits was given the same oral dose and concentration of recombinant L.lactis MG1363/pMG36e-p30-LTB-His,MG1363/pMG36e-p54-LTB-His MG1363/pMG36e-p72-LTB-His,1 mL for each.There were two rounds of immunizations,with an interval of 14 d between the primary immunization and the secondary immunization.Each immunization lasted for 3 d,once per day.Spleen cell survival rate,serum IL-4 and IFN-γcontents,IgG antibody level and s IgA content of small intestinal mucosa were detected by spleen cell,serum and small intestinal mucosa samples at 14 d after primary and secondary immunization respectively.The results showed that:(1)Splenic cell survival rate and serum IL-4and IFN-γcontents in groups 3 and 4 were significantly higher than those in groups 1 and 2(P<0.01),splenic cell survival rate in group 4 was significantly lower than that in group 3(P<0.01),IFN-γcontent was significantly higher than that in group 3(P<0.05),and IL-4 content was slightly higher than that in group 3(P>0.05).(2)Serum specific IgG antibodies were produced in groups 3 and 4,and the positive rates of p30-Ab,p54-Ab and p72-Ab were 80%on the 14th day after the primary immunization and 100%on the 14th day after the secondary immunization.(3)The s IgA content of small intestinal mucosa in groups 3 and 4 was significantly higher than that in groups 1 and 2(P<0.01),and that in group 4 was significantly higher than that in group 3(P<0.05).(4)There was no significant difference in average body weight among all groups on day 14 after the primary exemption(P>0.05);On the 14th day after the secondary exemption,the average body weight of groups 3 and 4 was significantly higher than that of groups 1 and 2(P<0.05),but there was no significant difference between groups 3 and 4(P>0.05).Conclusion:Recombinant L.lactis was successfully constructed in this experiment MG1363/pMG36e-p30-His,MG1363/pMG36e-p54-His,MG1363/pMG36e-p72-His and MG1363/pMG36e-p30-LTB-His,MG1363/pMG36e-p54-LTB-His,MG1363/pMG36e-p72-LTB-His produced an ideal immune response;Three strains of recombinant L.lactis expressing the target proteins showed ideal effect on promoting humoral immunity and Th1 cell immunity;Three strains of recombinant L.lactis expressing fusion proteins had good effects on promoting local mucosal immunity,humoral immunity and Th2 cell immunity. |
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