Recombinant Lactobacillus casei Expressing Capsid Protein VP60 can Serve as Vaccine Against Rabbit Hemorrhagic Disease Virus in Rabbits

Wang, Li; Xia, Tian; Guo, Tiantian; Ru, Yi; Jiang, Yanping; Cui, Wen; Zhou, Han; Qiao, Xinyuan; Tang, Lijie; Xu, Yigang; Li, Yijing

The main disadvantage of traditional plasmid expression systems is using antibiotic resistance genes as selective markers for genetically engineered bacteria [53, 54] . Although antibiotics are helpful for vector screening, using antibiotic resistance genes in practice can impact biosafety, contribute to antibiotic resistance, and present potential hazards to health and the environment [55] . To avoid potential issues caused by antibiotic resistance markers, it is important to establish expression vectors without the use of antibiotic resistance genes. eGFP has many advantages as a genetic marker and reporter gene. eGFP expression is mostly non-toxic to cells, and eGFP fluorescence can last for an extended time [55] . In the present study, we constructed a recombinant L. casei pPG-T7g10-eGFP-VP60/LC393 using eGFP as a selection marker and removed chloramphenicol resistance from the vector. Analysis of inherited stability of pPG-T7g10-eGFP-VP60/LC393 demonstrated that recombinant L. casei was stably inherited for more than 40 generations with no gene deletion. We designed the pPG-T7g10-PPT expression vector, a constitutive cell-surface expressed plasmid, to construct recombinant L. casei for the delivery of vaccine antigen. The pPG-T7g10-PPT vector contained a T7g10 enhancer, derived from gene 10 of bacteriophage T7; the T7g10 enhancer can augment protein translation and expression [56] . Expression of the eGFP-VP60(VP1) fusion protein was confirmed via western blotting and IFA. Our results indicate that a genetically engineered oral vaccine against RDHV, using L. casei without antibiotic resistance gene, was developed successfully.