Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.
Bacterial Proteins/metabolism* ; Bacterial Toxins/metabolism* ; Caco-2 Cells ; Clostridioides ; Clostridioides difficile/genetics* ; Humans ; Oxidoreductases/metabolism* ; Transcriptome ; Vaccines, Attenuated

OBJECTIVETo construct a recombinant Lactobacillus acidophilus that expresses high levels of Helicobacter pylori (Hp) adhesin Hp0410.

METHODSThe gene fragment encoding Hp0410 was amplified by PCR from the DNA of H. pylori NCTC11639 strain and cloned into the shuttle plasmid pMG36e to construct pMG36e-Hp0410, which was transformed into Lactobacillus acidophilus by electroporation. The target protein was confirmed with SDS-PAGE and silver nitrate staining and analyzed by Western blotting. The stability of the recombinant plasmid was assessed by drawing the growth curve of the recombinant Lactobacillus acidophilus.

RESULTSA 750-bp fragment was inserted into the pMG36e plasmid and transformed into Lactobacillus lactis. The transformed bacterium expressed the target protein with a relative molecular mass of about 34 kD. Western blotting confirmed that the expressed proteins could be recognized by the serum of patients with Hp infection. The recombinant plasmid pMG36e-Hp0410 exhibited good stability in the presence or absence of erythromycin.

CONCLUSIONSThe recombinant Lactobacillus acidophilus with high constitutive expression of Hp0410 has been constructed successfully.

Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Bacterial Vaccines ; biosynthesis ; Helicobacter Infections ; prevention & control ; Humans ; Lactobacillus acidophilus ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Attenuated ; biosynthesis

This study aimed to construct full-length cDNA clones of the Japanese encephalitis virus (JEV). SA14-14-2 strain and discuss the feasibility of constructing chimeric viruses for exogenous gene expression based on the JEV genetic skeleton. Long-fragment RT-PCR techniques were applied to amplify JEV cD-NAs, and two amplified fragments with corresponding restriction endonuclease sites at both ends were cloned into the pACYC184 vector sequentially. Using standard molecular techniques, the enhanced green fluorescent protein (EGFP) gene was inserted into the 3' non-coding region of JEV as a reporter gene. After in vitro transcription and transfection procedures, wild-type JEV and chimeric JEV that expressed the EGFP as the reporter gene were successfully rescued. The recovered viruses were characterized by RT-PCR, plaque assays, and direct fluorescence microscopy. After six serial passage generations, the stability of the recovered viruses were studied in terms of virus growth characteristics and structural gene expression. The results showed that cDNA clones of rJEV and rJEV-EGFP were successfully constructed and rescued in BHK-21 cells after in vitro transcription and transfection. Each generation of the recovered viruses was stable and the chimeric virus rJEV-EGFP could stably express EGFP. The findings of this study indicate that both rJEV and rJEV-EGFP could be constructed and rescued in BHK-21 cells, and the JEV SA14-14-2 strain could be obtained as a viral vector to express foreign genes.
Cloning, Molecular ; DNA, Complementary ; genetics ; metabolism ; Encephalitis Virus, Japanese ; genetics ; metabolism ; Encephalitis, Japanese ; virology ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Vaccines, Attenuated ; genetics ; metabolism ; Viral Vaccines ; genetics ; metabolism

The Yellow Fever (YF) vaccine, an attenuated yellow fever 17D (YF-17D) live vaccine, is one of the most effective and safest vaccines in the world and is regarded as one of the best candidates for viral expression vector. We here first reported in China the construction and characterization of the recombinant expression vector of yellow fever 17D which contained the proteinase 2A fragment of foot-and-mouth disease virus (FMDV). Three cDNA fragments representing the full-length YF-17D genome, named 5'-end cDNA (A), 3'-end cDNA (B) and middle cDNA (C), were obtained by reverse transcription polymerase chain reaction (RT-PCR), together with the introduction of SP6 enhancer, necessary restriction sites and overlaps for homologous recombination in yeast. Fragment A and B were then introduced into pRS424 in turn by DNA recombination, followed by transfection of fragment C and the recombinant pRS424 containing A and B (pRS-A-B) into yeast. A recombinant vector containing full length cDNA of YF-17D (pRS-YF) was obtained by screening on medium lack of tryptophan and uracil. A recombinant YF-17D expression vector containing FMDV-2A gene fragment (pRS-YF-2A1) was then constructed by methods of DNA recombination and homologous recombination in yeast described above. In vitro transcription of the recombinant vector pRS-YF-2A1 was then carried out and introduced into BHK-21 cells by electroporation. Results of indirect immunofluorescence assay (IFA) and titer determination showed a stable infectious recombinant virus was gotten, whose features such as growth curve were similar to those of the parental YF-17D. The results suggest that the recombinant vector pRS-YF-2A1, by introduction of heterogenous genes via 2A region, is potential to be an effective live vaccine expression vector.
Animals ; Cell Line ; Cloning, Molecular ; Cricetinae ; Epitopes ; immunology ; Foot-and-Mouth Disease ; prevention & control ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Genetic Engineering ; Genetic Vectors ; Recombination, Genetic ; Saccharomyces cerevisiae ; genetics ; metabolism ; Vaccines, Attenuated ; Viral Vaccines ; genetics ; immunology ; Yellow fever virus ; genetics ; immunology

Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high-growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6 + 2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.
Animals ; Chick Embryo ; Chickens ; Genetic Engineering ; Hemagglutinins, Viral/genetics/metabolism ; Humans ; Influenza A Virus, H5N1 Subtype/*genetics/immunology ; Influenza A Virus, H9N2 Subtype/*genetics/immunology ; Influenza Vaccines/*genetics/metabolism ; Influenza in Birds/immunology/virology ; Influenza, Human/immunology/*prevention & control/virology ; Neuraminidase/genetics/metabolism ; Transgenes ; Vaccines, Attenuated/*genetics/metabolism ; Viral Proteins/genetics/metabolism

OBJECTIVETo prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection.

METHODSUsing genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected.

RESULTSThe sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed.

CONCLUSIONThe attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; administration & dosage ; immunology ; Female ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Helicobacter Infections ; immunology ; prevention & control ; Helicobacter pylori ; enzymology ; genetics ; immunology ; Immunoglobulin G ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Mice, Inbred BALB C ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Urease ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; genetics ; immunology ; Weight Loss

OBJECTIVETo study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMV-beta as report gene.

METHODSMurine peritoneal macrophages were infected with SL7207(pCMV-beta) in vitro, then the expression of the beta-gal were detected by X-gal staining or RT-PCR. After mice were orally immunized with SL7207(pCMV-beta), the expression of beta-gal in the lymphoid tissue were tested by RT-PCR, humoral responses were tested by ELISA, splenic lymphocyte proliferation were tested by 3H-TdR incorporation and cytotoxic T lymphocyte reaction were tested by JAM test.

RESULTSThe results indicated that the plasmid pCMV-beta could be delivered by SL7207 into the nucleus of the murine macrophages efficiently and expressed well in vitro; after mice received oral immunizations with attenuated S.typhimurium SL7207 harboring plasmid pCMV-beta mice, the expression of beta-gal could be detected in the spleen, mesenteric lymph nodes and Peyers patches of the mice. Furthermore, the experiments demonstrated that specific humoral immune responses and cell-mediated immune responses were successfully induced in these immunized mice. Compared with the naked DNA vaccination, SL7207 (pCMV-beta) oral immunization were more efficient in inducing cellular immune responses.

CONCLUSIONSAttenuated Salmonella typhimurium SL7207 could be used as DNA delivery vehicle for oral immunization, which have the ability to deliver the antigen-encoding DNA specifically to APC directly for inducing the specific immune response being dominant with cellular immune response.

Animals ; Bacterial Vaccines ; immunology ; Cell Proliferation ; Cytotoxicity, Immunologic ; Female ; Genes, Reporter ; Genetic Vectors ; Macrophages, Peritoneal ; metabolism ; microbiology ; Mice ; Mice, Inbred BALB C ; Mutation ; Salmonella typhimurium ; genetics ; T-Lymphocytes, Cytotoxic ; cytology ; Transfection ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; beta-Galactosidase ; genetics ; immunology ; metabolism

OBJECTIVETo evaluate the feasibility of oral cytokine gene therapy against tumor using live attenuated Salmonella as a vector.

METHODSA live attenuated AraA- autotrophic mutant of Salmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1 and pCMVmIL-12 administered orally in BALB/c and C57BL/6 mice. After 6 weeks, the mice were challenged with 4T1 or Lewis tumor cells, respectively, and flow cytometry and confocal microscopy were used to detect the expression of green fluorescence protein (GFP) in the tissues. PCR and ELISA were performed to detect the integration and expression of mIL-12 gene, and the survival time of the mice was also recorded.

RESULTSGFP expression and mIL-12 gene integration could be detected in the liver, spleen, intestinal, kidney and tumor tissues of the mice. The serum level of mIFN-gamma, mIL-12 increased significantly in mice with oral mIL-12 administration (P<0.05), which resulted in the survival prolongation of the mice as compared with the control mice (P<0.05).

CONCLUSIONOral gene therapy using live attenuated Salmonella can be potentially a simple, effective and above all, safe means for tumor treatment.

Administration, Oral ; Animals ; Carcinoma, Lewis Lung ; therapy ; Flow Cytometry ; Genetic Therapy ; methods ; Genetic Vectors ; administration & dosage ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Interleukin-12 ; genetics ; metabolism ; Mammary Neoplasms, Animal ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Microscopy, Confocal ; Neoplasms, Experimental ; therapy ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; immunology