title: 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.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China.
authors.forename: Ji-Yuan
authors.initials: JY
authors.lastname: Yin
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China.
authors.forename: Chao-Qun
authors.initials: CQ
authors.lastname: Guo
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China.
authors.forename: Zi
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China.
authors.forename: Mei-Ling
authors.initials: ML
authors.lastname: Yu
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China.
authors.forename: Syed M
authors.initials: SM
authors.lastname: Bukhari
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China.
authors.forename: Li-Jie
authors.initials: LJ
authors.lastname: Tang
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China. yigangxu_china@sohu.com.
authors.forename: Yi-Gang
authors.initials: YG
authors.lastname: Xu
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street No. 59, Harbin, Xiang Fang District, China. yijingli@163.com.
authors.forename: Yi-Jing
authors.initials: YJ
authors.lastname: Li
mesh_terms: D000284:Administration, Oral; D000818:Animals; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D042822:Genomic Instability; D007780:Lacticaseibacillus casei / Q000235:genetics*; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D022243:Rotavirus Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins; D022243:Rotavirus Vaccines; C060339:VP4 protein, Rotavirus; D014614:Vaccines, Synthetic
keywords: Immunogenicity analysis; Lactobacillus casei; Markerless integration; Microarray analysis; VP4 genome expression