Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393

Abstract

Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.

Immunomodulatory Properties of Bacterium-Like Particles Obtained From Immunobiotic Lactobacilli: Prospects for Their Use as Mucosal Adjuvants

“…By using a directed chromosomal integration approach, Yin et al (28) were able to develop a stable L. casei strain expressing the porcine rotavirus VP4 antigen. The oral immunization of mice with recombinant L. casei VP4 induced both specific intestinal and systemic humoral immune responses.…”

Oral Immunization of Chickens with Probiotic Lactobacillus crispatus Constitutively Expressing the α-β2-ε-β1 Toxoids to Induce Protective Immunity

“…Specific IgA antibodies can interact with secretory fragments produced by the intestinal mucosal epithelium on the surface of the intestinal mucosa to prevent adhesion of pathogenic bacteria and invasion of mucosal tissues [ 15 , 16 ]. Specific lymphocytes then dissociate in the mucosal lamina propria, glands, and other reactive sites to trigger an immune response at multiple mucosal sites [ 17 ]. With continuous improvements in research technology, it is now possible to obtain genetically engineered Lactobacillus with better antigenic quality, whose application in production practices has far-reaching significance.…”

Recombinant Lactococcus lactis Expressing Grass Carp Reovirus VP6 Induces Mucosal Immunity Against Grass Carp Reovirus Infection

“…Amplicons were subcloned into plasmid pNZ8148 (Novagen, Madison, WI, USA) using Nco I and Xba I restriction enzymes resulting in recombinant pNZ8148- VP6 . The recombinant plasmid was introduced into L. lactis NZ9000 by electroporation as previously described ( 23 ). The L. lactis transformants were cultured in M17 agar containing 5 μg/mL chloramphenicol and incubated at 30°C for 24 h. Positive clones were selected and confirmed by PCR using VP6 primers ( Table 1 ).…”

“…Protein expression was induced by the addition of using nisin (Weijia, Guangzhou, China) for 4 h. L. lactis cells were then collected by centrifugation and the pellets were suspended in PBS. The cells were lysed with the addition of 10 mg lysozyme and incubation at 37°C for 1 h. The cell lysates were then further processed as described previously ( 23 ). Protein production was analyzed using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA).…”

“…To test the specific IgM levels against VP6 in the antisera of immunized fish, anti-IgM MAbs were prepared and kept in our laboratory. Serum antibody levels were determined by ELISA as described previously ( 23 ). Briefly, recombinant VP6 (rVP6) ( 24 ) (8 μg/mL in carbonate-bicarbonate buffer) was used as antigen to coat wells overnight at 4°C.…”