Construction And Immunogenicity Of Recombinant Lactic Acid Bacteria Expressing Cap Protein Of PCV3

Porcine circovirus(PCV)is a single-stranded circular DNA virus,its belong to the Circovirus of the Circoviridae family.Currently identified genotypes are PCV1、PCV2 and PCV3.In 2016,Phan TG et al revealed PCV3 by the Metagenome Sequencing,which was clinically manifested porcine dermatitis and nephropathy syndrome-like、reproduction disorders and multisystem inflammation.With the increasing of large-scale and modernized breed industry,viral infectious diseases such as PCV3 have become more and more serious,and the difficulty in prevention and control has been increasing.The need for a safe and effective new vaccine is increasing.PCV3 Cap protein is the only structural protein and the strongest immunogenicity of PCV3.In this study,the optimized PCV3 Cap gene was cloned,and a recombinant expression plasmid was constructed and tested in the intermediate host Lactococcus NZ3900 and expressed in the terminal host Lactobacillus plantarum Lp12.Finally,C57BL/6 mice were immunized with recombinant Lactobacillus plantarum and the mucosal immune protective effect was detected.1.Lactobacillus recombinant expression vector molecule design and constructionThe PCV3 Cap gene contained signal peptides 1320 and DCpep(Dendritic cells peptide)and codon optimized by Lactobacillus plantarum was synthesized.The gene was ligated to the expression vector pSIP411 by PCR amplification and seamless cloning technology.Then it was transferred to the intermediate clone host NZ3900 and screened positive clones by PCR and sequencing.After that the positive clone was retransmitted into Lactobacillus plantarum Lp12 for induced expression.The expression products were confirmed by western blot,indirect immunofluorescence and flow cytometry.The positive rate was 47.9%.Indirect immunofluorescence further confirmed the expression of antigen on the surface of bacteria.2.Optimization of PCV3 Cap protein expression by recombinant Lactobacillus plantarumLactic acid bacteria expression system has potential application value in the future because of its probiotic effect and immune adjuvant effect.However,compared with the prokaryotic expression system,its expression level needs to be increased.The expression condition was optimized in this study.The results shown that highest expression was at 75-125 ng/mL concentration of SppIP inducer.There was a little difference in expression at different temperatures.The expression was highest with the induce for 8 hours.The stable expression was achieved at 96 hours at room temperature and in 15 passages.3.Probiotic characteristics of Lactobacillus plantarum and recombinant Lactobacillus plantarumIn order to verify whether the modified Lactobacillus plantarum has changed its own characteristics,this study measured its bacterial body and colony characteristics,growth characteristics,acid and bile salt resistance and stress resistance,adhesion and DCs cell antigen presentation ability.By comparison,the results showed that the characteristics of recombinant Lactobacillus plantarum did not change.4.Immunization of C57BL/6 mice with recombinant Lactobacillus plantarum and detection of immune effectIn this study,C57BL/6 mice were divided into 3 groups(negative control group:PBS,Lp 12 group,and Lp 12:pSIP411-PCV3D group).Immunization was performed by gavage.After the immunization,mouse stool,serum samples,and intestinal lavage fluid were collected.ELISA was used to detect the expression level of spleen lymphocyte subsets by flow cytometry CCK-8 was used to detect the increase of spleen lymphocytes.The results showed that in the CD4+T lymphocyte response,Lp12 group and Lp12:pSIP411-PCV3D group showed a slight increase compared with the PBS group,but no statistical difference.CCK-8 test results showed that Lp12 and Lp12:pSIP411-PCV3D has no statistical difference compared with PBS.The intestinal lavage fluid and total stool SIgA test indicated that Lp12 group and Lp12:pSIP411-PCV3D group showed a downward trend,and the overall trend in feces also showed a decrease,but no statistical difference,compared with the PBS group.The total IgG2a level in the antibody level was significantly increased,indicating that the level of cellular immunity was enhanced.Tthe changes of Th1,Th2,and Th17 cytokines in the experimental group were higher than those in PBS and pSIP411-PCV3D,Lp12 control group.The results shown that Cap protein can be well expressed in Lactobacillus plantarum Lp12.Our results suggested that the recombinant PCV3 Cap protein has a some effect of activating T cell-mediated immunity,but the mechanism is still worthy of further exploration.