The Surface Of Lactobacillus Casei Expressing The VP2from IBDV And Research Of Immunogenicity

In this study, we constructed Lactobacillus casei expressing VP2of infectious bursal diseasevirus (IBDV). The recombinanted Lactobacillus casei could induce mucosal immune andsystem immune by oral administration.First of all, primers were employto amplify the cDNA ofthe VP2gene by polymerase chain reaction (PCR).The target gene were built on the vector pLA,resulting in recombinant plasmid pLA-VP2after verified by PCR.Then the recombinantplasmid pLA-VP2was directly used to transform DH5α and cultured in LB with ampicillinmedium. Plasmid DNA was extracted and established in Lactobacillus casei by electroporation.After verificated, we achieved the pLA-VP2/L. Casei. The recombinant strains were induce toexpress VP2protein, which was detected by SDS-PAGE, Western Blot and flow cytometry.SDS-PAGE and Western Blot results showed that a molecular weight of about90kDa weredetected, and recombinant VP2protein could bind to IBDV positive serum. The light scatteringdetector of flow cytometry could capture the fluorescence of recombinant strains on the surfaceof the cell wall, and the fluorescence signal was stronger than control.The expression efficiencyof fusion protein pgsA-VP2was80%as normal distribution curve. In conclusion, VP2proteinfrom IBDV had expressed on the surface of Lactobacillus casei already.In order to determine immunization times and dosage, to evaluate immunogenicity andprotective effect in chicks, we used the Lactobacillus casei (L. casei) anchoring the VP2proteinof infectious bursal disease virus (IBDV) as antigen transfer system. Firstly, the five-day-oldchicks divided into two groups (twice and three times inoculation group, respectively) were fedwith pLA-VP2/L. casei at three dosages of108,109, or1010CFU/mL. The level of serum IgGand sIgA in small intestine washing fluid was detected by indirect ELISA. The immunizedchicks and control chicks were challenged by IBDV/DQ at lethal dose to determine theprotective ratio at day7after the last immunization. Secondly, according to the determinedimmunization times and dosage, we vaccinated twice with109CFU/mL per chick inpLA-VP2/L. casei oral group, pLA-VP2/L. casei intranasal group and pLA/L. casei oralgroup(control); chicks in commercial vaccine oral group and injection group were immunizedaccording to the vaccine instruction and orally administrated in PBS group as control. IndirectELISA monitored the specific anti-VP2serum IgG and sIgA antibodies in relevant mucosal sites. Splenocyte proliferation assay was investigated by MTT assay at day7after the lastimmunization, and at the same time the chicks were challenged by IBDV/DQ. The degree ofinjury in bursa of Fabricius was observed and the lesion score was recorded at day7postchallenge. Data showed that immunization times and dosage was twice and109CFU/mL,respectively. The results of immune route experiments indicated that the level of specific IgGand sIgA in each of pLA-VP2/L. casei and commercial vaccine groups was significantly higherthan that in pLA/L. casei and PBS control groups (P<0.01) and protection efficacy inpLA-VP2/L. casei oral group was higher than that in pLA-VP2/L. casei intranasal group.Splenocyte proliferation assay showed the stimulation index (S.I.) of pLA-VP2/L. casei oralgroup was significant higher than the other groups (P<0.01). The lesion score indicated thepLA-VP2/L. casei was safer than commercial vaccine for bursa of Fabricius. These resultsdemonstrate that the pLA-VP2/L. casei could be a vaccine candidate for IBDV.