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

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.

Chromosomal insertions in the Lactobacillus casei upp gene that are useful for vaccine expression.

To develop a stable and marker-free Lactobacillus strain useful for the expression of vaccines, we developed a temperature-sensitive suicide plasmid with expression cassettes containing an HCE promoter, a PgsA anchor, the alpha-toxin gene, and an rrnB T1T2 terminator (PPαT) that uses a 5-fluorouracil (5-FU) counterselectable marker for Lactobacillus casei. Three strains containing the correct PPαT expression cassettes were produced via the selective pressure of 5-FU screening. We confirmed that the upp gene was deleted and that the PPαT expression cassettes were inserted into the upp site of L. casei ATCC 393 by genomic PCR amplification and sequencing. 5-FU resistance in recombinant bacteria could be stably inherited for as long as 40 generations following insertion. However, bacteria containing the integrated DNA grew more slowly than wild-type L. casei. An indirect enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that the alpha-toxin gene was expressed. Also, we visualized expression of the protein on the surface of L. casei cells using laser confocal microscopy. These results taken together demonstrate that these recombinant bacteria should provide a safe tool for effective vaccine production.

Oral vaccination with the porcine rotavirus VP4 outer capsid protein expressed by Lactococcus lactis induces specific antibody production.

The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

Efficacy and safety of acupuncture in patients with cancer-related fatigue: A protocol for systematic review and meta-analysis.

BACKGROUND: Cancer-related fatigue (CRF) is considered a common complication of cancer or cancer treatment, which has a serious adverse effect on the life and of cancer patients, leading to a decline in their quality of life (QoL). The existing clinical trials revealed that acupuncture has a positive effect on CRF, and there are fewer adverse events confirmed in the corresponding systematic review. However, in recent years, new studies on using acupuncture to treat CRF were conducted, so in order to evaluate its efficacy, an updated systematic review. This protocol provides research methods for systematic review and meta-analysis of the safety and effectiveness of acupuncture for the treatment of CRF. METHODS: We will searched the randomized controlled trial literature of acupuncture treatment for CRF in 4 English and 4 Chinese databases, including PubMed, MEDLINE, Web of Science, Cochrane Central Register of Controlled Trials (Central), China National Knowledge Infrastructure (CNKI), China Biomedical Literature Database(CBM), China Science Journal Database (VIP), and Wanfang Database. Simultaneously, other resources are manually retrieved which include reference lists of identified publications, conference articles, and grey literature. We also included the clinical randomized controlled trials of acupuncture treatment for CRF in the study. The search language is limited to Chinese and English. Two trained reviewers independently completed research screening, data extraction, and research quality assessment. RevMan (V.5.3) software was used to perform data statistical analysis and Grading of Recommendations Assessment, Development, and Evaluation (GRADE) was used to evaluate the quality of evidence. RESULTS: This study is based on past and present clinical evidence to comprehensively evaluate the effectiveness and safety of acupuncture treatment for CRF. CONCLUSION: Through this systematic review, we will provide the latest high-quality evidence of whether acupuncture treatment for CRF is effective and safe and also provide a theoretical basis for clinicians to choose acupuncture for the treatment of CRF. SYSTEMATIC REVIEW REGISTRATION: INPLASY 202090049.

[Progress of the mechanism research of acupuncture in treatment of diet-induced obesity].

The literature of experimental research on diet-induced obesity treated with acupuncture was retrieved. The aspects of epigenetics, neuroendocrine system, intestinal flora, inflammatory response, oxidative stress and substance metabolism of the mechanism of acupuncture in the treatment of diet-induced obesity were summarized. It is suggested that the potential mechanism of acupuncture in the treatment of diet-induced obesity should be discussed in the aspects of the interaction of Toll-like receptors on obesity-intestinal flora-immune function, the improvement of insulin resistance, epigenetics and antioxidant stress.

Immunogenicity in chickens with orally administered recombinant chicken-borne Lactobacillus saerimneri expressing FimA and OmpC antigen of O78 avian pathogenic Escherichia coli.

PURPOSE: Avian colibacillosis is responsible for economic losses to poultry producers worldwide. To combat this, we aimed to develop an effective oral vaccine for chicken against O78 avian pathogenic Escherichia coli (APEC) infection through a Lactobacillus delivery system. METHODOLOGY: Eight Lactobacillus strains isolated from the intestines of broiler chickens were evaluated based on their in vitro adherence ability to assess their potential as a delivery vector. Fimbrial subunit A (FimA) and outer-membrane protein C (OmpC) of APEC with and without fusion to dendritic cell-targeting peptide (DCpep) and microfold cell-targeting peptide (Co1) were displayed on the surface of Lactobacillus saerimneri M-11 and yielded vaccine groups (pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, respectively). The colonization of the recombinant strains in vivo was assessed and the immunogenicity and protective efficacy of orally administered recombinant strains in chickens were evaluated. RESULTS: The colonization of the recombinant strains in vivo revealed no significant differences between the recombinant and wild-type strains. Chickens orally administered with vaccine groups showed significantly higher levels of OmpC/FimA-specific IgG in serum and mucosal IgA in cecum lavage, nasal lavage and stool compared to the pPG/M-11 group. After challenge with APEC CVCC1553, better protective efficacy was observed in chickens orally immunized with pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, but no significant differences were observed between the two groups. CONCLUSIONS: Recombinant chicken-borne L. saerimneri M-11 showed good immunogenicity in chickens, suggesting that it may be a promising vaccine candidate against APEC infections. However, the activity of mammalian DCpep and Co1 was not significant in chickens.

[Construction of recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis spike glycoprotein and analysis of immunogenicity].

Transmissible gastroenteritis virus (TGEV), is an enteropathogenic coronavirus that causes a highly fatal acute diarrhea in newborn pigs. It's typically clinical manifestations consist of omitting, severe diarrhea, loss water and highly infectious disease. All kinds and ages of pigs can be infected. Particular, the mortality piglets under 3 weeks may reach 100% . The effective protection against TGEV requires the development of vaccines that are able to induce local mucosal immunization. Lactococcus lactis was selected as a bacterial carrier for the expression of TGEV spike glycoprotein. The gene of S glycoprotein was cloned into the Lactococcus lactis vectors pNZ8112. An approximately 2000 bps fragments of TGEV gene S that encompasses all the four major antigenic domains critical for neutralization was transformed into Lactococcus lactis NZ9000 by electroporation, resulting in the recombinant strain pNZ8112-Sa/NZ9000. The recombinant glycoprotein S was detected by SDS-PAGE and Western blot after induced by 1ng/mL nisin. The result indicated that the expressed product maintain the antigenicity of TGEV as expected. In order to detect the location of expressed protein, the yellow and green fluorescence of the recombinant strain pNZ8112-Sa/NZ9000 was detected by the IFA experiments, which indicated that the expressed recombinant protein was secreted and located on the surface of the bacterium cell. Oral immunization of BALB/c mice with recombinant strain that constitutively express the 66kDa fragment of the glycoprotein S, Specific anti-TGEV glycoprotein S secret immunoglobulin A (sIgA) antibodies were detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces after immunization. It was showed that the mice immunized with pNZ8112-Sa/NZ9000 recombinant strain had produced clear antibody level anti TGEV, and which had provided important substance foundation and explored the feasibility of Lactobacillus as oral vaccine.

Use of dual priming oligonucleotide system-based multiplex RT-PCR combined with high performance liquid chromatography assay for simultaneous detection of five enteric viruses associated with acute enteritis.

In this study, a specific and sensitive method for simultaneous detection of human astrovirus, human rotavirus, norovirus, sapovirus and enteric adenovirus associated with acute enteritis was developed, based on the specific dual priming oligonucleotide (DPO) system and the sensitive high-performance liquid chromatography (HPLC) analysis. The DPO system-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) combined with HPLC assay was more sensitive than agarose gel electrophoresis analysis and real-time SYBR Green PCR assay, and showed a specificity of 100% and sensitivity of 96%-100%. The high sensitivity and specificity of the assay indicates its great potential to be a useful tool for the accurate diagnosis of enteric virus infections.

Development of a loop-mediated isothermal amplification assay for rapid detection of bovine parvovirus.

A loop-mediated isothermal amplification (LAMP) assay was developed for detection of bovine parvovirus (BPV) DNA. Four primers were designed to recognize six distinct regions on the target DNA based on a highly conserved sequence in the VP2 region of the BPV genome. The optimized LAMP reaction conditions were 8 mM Mg²âº, 1.2 mM betaine, and an incubation at 63°C for 45 min. After amplification the products were detected either by observing a ladder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. The detection limit of the LAMP assay was 9 copies of BPV-DNA and was 100 times more sensitive than conventional PCR. A ladder pattern of bands after gel electrophoresis was observed for only BPV isolates and showed that the BPV LAMP assay was highly specific without any cross-reactivity with other related viruses. The LAMP assay was evaluated further using 59 field samples and the results were comparable to conventional PCR. The LAMP assay is a simple, rapid and economic detection method; it can provide a useful technique suitable for detection of BPV infection in both field conditions and laboratory settings.

[The surface display of porcine parvovirus VP2 protein in Lactobacillus casei].

Lactobacillus casei 393 was selected as a bacterial carrier for the expression of Porcine Parvovirus (PPV) protective antigen VP2 protein. The gene encoding PPV VP2 protein was cloned into the Lactobacillus casei surface expression vector pPG, and then the constructed recombinant vector pPG-VP2 was electrotransformed into Lactobacillus casei 393 generating the recombinant system pPG-VP2/L. casei393 expressing PPV VP2 protein. The recombinant strain was induced by 2% Lactose in MRS and about 74kD protein was detected with SDS-PAGE. The result of Western-blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescent test showed that the expressed protein was secreted on the cell surface Lactobacillus casei.