The synthetic PDCoV N protein gene was optimized and cloned into the pET-30a vector to obtain the pET-30a-N plasmid.Thenthe recombinant plasmid was transformed into three strains of BL21 E.coli using heat-shock to explore protein expression conditions.The expressed proteins was purified using Ni Focurose 6FF(IMAC)and used as antigen to immunize the New Zealand White rabbit to prepare the polyclonal antibody against the PDCoV N protein.The antibody titer was measured by indirect ELISA method.The specificity for the antibody was identified by West-ern blot and indirect immunofluorescence(IFA).The results showed that the pET-30a-N plasmid showed high expression level in BL21 StarTM(DE 3).The optimal expression condition was 37 ℃ 4 h.The purity of the target protein could reach 90.3％after purification.Indirect ELISA showed that the antibody titers was up to 1∶204 800.Western blot and IFA showed that the produced rabbit polyclonal antibody exhibited good specificity.In conclusion,the polyclonal antibody was prepared which specifically recognized the PDCoV N proteins.The results provided some references for the subsequent exploration of PDCoV N protein function and laid a foundation for establishing a diag-nostic method for PDCoV.

The synthetic PDCoV N protein gene was optimized and cloned into the pET-30a vector to obtain the pET-30a-N plasmid.Thenthe recombinant plasmid was transformed into three strains of BL21 E.coli using heat-shock to explore protein expression conditions.The expressed proteins was purified using Ni Focurose 6FF(IMAC)and used as antigen to immunize the New Zealand White rabbit to prepare the polyclonal antibody against the PDCoV N protein.The antibody titer was measured by indirect ELISA method.The specificity for the antibody was identified by West-ern blot and indirect immunofluorescence(IFA).The results showed that the pET-30a-N plasmid showed high expression level in BL21 StarTM(DE 3).The optimal expression condition was 37 ℃ 4 h.The purity of the target protein could reach 90.3％after purification.Indirect ELISA showed that the antibody titers was up to 1∶204 800.Western blot and IFA showed that the produced rabbit polyclonal antibody exhibited good specificity.In conclusion,the polyclonal antibody was prepared which specifically recognized the PDCoV N proteins.The results provided some references for the subsequent exploration of PDCoV N protein function and laid a foundation for establishing a diag-nostic method for PDCoV.

This study aims to investigate the effect of Lactiplantibacillus plantarum LP12 on obe-sity prevention.In our study,Lactiplantibacillus plantarum LP12 was added to the diet for feed-ing,and the blood biochemistry status of rabbit,as well as the antioxidant effect of serum and liver samples were analyzed by determining the body weight change and feed intake of Japanese White rabbits.The changes in colony structure and abundance were also analyzed by 16S rDNA sequen-cing.The results showed that supplementation of Lactiplantibacillus plantarum LP12 inhibits weight gain,decreases serum glucose and ALT levels,and increases SOD activity in the liver.16S RNA gene sequencing analysis showed that the addition of Lactiplantibacillus plantarum LP12 increases the abundance of Bacteroidetes and Desulfovibrioides at the phylum level,and the supple-mentation of Lactiplantibacillus plantarum LP12 increases the abundance of Muribaculaceae at the genus level.Predictive analysis of microbiota function revealed that the supplementation of Lactiplantibacillus plantarum LP12 positively regulated iron-sulfur clusters and Zn-dependent proteases.In conclusion,the addition of Lactiplantibacillus plantarum effectively inhibits weight gain in Japanese White rabbits,enhances the antioxidative activity of the liver,and induces altera-tions in the gut microbiota composition of these rabbits.These findings lay an experimental foun-dation for further exploring the mechanisms by which Lactobacillus plantarum LP12 exerts its preventive effects against obesity and promotes metabolic health.

This study aims to investigate the effect of Lactiplantibacillus plantarum LP12 on obe-sity prevention.In our study,Lactiplantibacillus plantarum LP12 was added to the diet for feed-ing,and the blood biochemistry status of rabbit,as well as the antioxidant effect of serum and liver samples were analyzed by determining the body weight change and feed intake of Japanese White rabbits.The changes in colony structure and abundance were also analyzed by 16S rDNA sequen-cing.The results showed that supplementation of Lactiplantibacillus plantarum LP12 inhibits weight gain,decreases serum glucose and ALT levels,and increases SOD activity in the liver.16S RNA gene sequencing analysis showed that the addition of Lactiplantibacillus plantarum LP12 increases the abundance of Bacteroidetes and Desulfovibrioides at the phylum level,and the supple-mentation of Lactiplantibacillus plantarum LP12 increases the abundance of Muribaculaceae at the genus level.Predictive analysis of microbiota function revealed that the supplementation of Lactiplantibacillus plantarum LP12 positively regulated iron-sulfur clusters and Zn-dependent proteases.In conclusion,the addition of Lactiplantibacillus plantarum effectively inhibits weight gain in Japanese White rabbits,enhances the antioxidative activity of the liver,and induces altera-tions in the gut microbiota composition of these rabbits.These findings lay an experimental foun-dation for further exploring the mechanisms by which Lactobacillus plantarum LP12 exerts its preventive effects against obesity and promotes metabolic health.

To study the biological characteristics and gene function of Ligilactobacillus salivarius CICC23174 strain,a strain isolated from chicken intestines.The next generation sequencing plat-form HiSeq2000 was used for sequencing,and bioinformatics software was employed to assemble and optimize the raw data.Functional annotation,bacteriocin and signal peptide,gene collinearity comparison,and phylogenetic analysis were performed on its gene information.Our results indica-ted that the linear genome of Ligilactobacillus salivarius CICC23174 was 203 542 bp in length,with a GC content of 32.84％,encoding 1 890 genes,containing 3 bacteriocins,and 50 signal pep-tides.Importantly,the Ligilactobacillus salivarius CICC23174 included into 27 unique genes,forming four systems:the Type Ⅰ CRISPR Cas defense system,the Type Ⅱ toxin antitoxin system,the tumor escaped inhibitory gene,and the protein secretion pathway.Moreover,collinearity analy-sis showed that CICC23174 strain was most similar to the Ligilactobacillus salivarius UCC118,but 16S rRNA phylogenetic analysis indicted that CICC23174 strain was genetically close to FZJTZ9M6 strain.The results of this study lay the foundation for enriching the species evolution-ary library of Lactobacillus salivarius and exploring the potential probiotic functions of Lactoba-cillus salivarius.

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the main pathogen that causes COVID-19,which is fast-mutating and highly transmissible.The infection has led to a global epidemic.As the main preventive and control measure,vaccination plays a critical role in fighting a-gainst COVID-19.Although a large number of epitope-based and mucosal vaccines have been stud-ied,few peptide epitope vaccines targeting the mucosa and their functional evaluation have been re-ported.In this study,we used SARS-CoV-2 structural protein M peptide epitope predicted by the IEDB database as an antigenic target to design the MS-3S gene containing 3 050 and 1 229 signal peptides and DCpep optimized for insertion into MS2 phage coat proteins.The expression plasmid pSIP:MS-3S was constructed by cloning the PCR fragments seamlessly and was transformed into Lactiplantibacillus plantarum 18 to obtain the recombinant bacterium LP18:MS-3S.Expression conditions such as induction time,inducer concentration,rotational speed and initial pH were opti-mized.The intranasal immunization experiments were performed to examine the vaccine efficacy.The results showed that the 916 bp-long target gene MS-3S modified and optimized was amplified and used to successfully construct the recombinant bacterial strain LP18:MS-3S.The optimal con-ditions for recombinant protein expression were obtained and verified by Western blot,flow cy-tometry,immunofluorescence and other detection methods.The optimal expression conditions were determined as follows:induction time was 4 h with 100 pg/L of SppIP as the optimal induction concentration.Antibody-specific for the epitope was verified by ELISA experiments in serum,alve-olar lavage fluid and fecal dilutions of mice.In summary,a recombinant bacterial strain expressing the epitope antigen of the SARS-CoV-2 M protein peptide was constructed.The obtained protein can induce the body to produce humoral and mucosal immunity,which lays the foundation for the development of a vaccine candidate for the mucosal immunity of COVID-19.