[Construction and Fluorescence Analysis of the Recombinant Listeria ivanovii Strain Expressing Green Fluorescent Protein].

OBJECTIVE: Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. METHODS: The promoter of Listeria monocytogenes Listeriolysin O (phly) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW-phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. RESULTS: The exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP. The recombinant plasmid pCW-phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. CONCLUSION: The prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.

[Prediction and Analysis of Epitopes in clpP2 of Mycobacterium tuberculosis].

OBJECTIVES: To predict and analyze the antigenic epitopes in Mycobacterium tuberculosis protein caseinolytic protease P2 (clpP2), and explore its possibility to be applied as a new tuberculosis (TB) vaccine and drug development target. METHODS: Secondary structure of clpP2 based on nucleic sequence was predicted by DNA Star software. The homologous sequence conformation were analyzed by Swiss-Model online software. T cells antigenic epitopes were predicted through VaxiPred, and B cell epitopes were predicted by combining use of several different prediction programs, such as ABCpred, COBEPro and BepiPredPred. The immune characteristics of clpP2 were analyzed by DNA Star, SignalP, TMHMM online software and were searched through NCBI database. RESULTS: clpP2protein was diverse in structure, composing with a great deal of CTL and Th cell epitopes. clpP2 was also predicted to comprise rich potential liner and discontinuous B-cell epitopes. These epitopes were accessible on the protein surface, located in flexible and hydrophilic regions. CONCLUSION: clpP2 is prompted to induce immune responses and developes a novel target in surveillance, treatment and vaccine.

[Genetic Recombiniation and Protein Expression Detection of Listeria-based tuberculosis Vaccine Candidates.]

OBJECTIVES: Genetic construction of tuberculosis vaccine candidates based on Listeria(L.) monocytogenes,L.ivanovii,and evaluation their protein expression,in order to provide a novel method for research on tuberculosis controlling. METHODS: Two kinds of gene cassettes carrying tuberculosis antigen encoding gene Rv3875 or Rv0129c were inserted into targeting vector harboring L.monocytogenes,L.ivanovii homologous sequences via genetic connection methods and plasmid transformation technology in vitro.Targeting plasmids were electroporated into L.monocytogenes,L.ivanovii,and the recombinant strains were experienced serial passage at 42 °C and 30 °C.Subsequently,the tuberculosis antigen gene cassettes in targeting plasmids were integrated into L.monocytogenes and L.ivanovii attenuated strain (knocking out of virulence gene actA and plcB) and L.ivanovii wild type strain by homologous recombination and gene targeting technology.The recombinant strains were screened by blue-white spot and antibiotic resistance test;the intracellular and extracellular proteins of the recombinant strains were tested by Western blot. RESULTS: Five recombination strains carried antigen gene cassette were constructed,and the recombinant genome were confirmed by PCR and sequencing.No erythromycin resistance gene was found in 5 strains,which was coincident to expection.Recombination strains Li-Rv0129c,Li-ΔactAplcB-Rv0129c and Li-ΔactAplcB-Rv3875 expressed Mycobacterium tuberculosis antigenic protein,Ag85C or ESAT-6,as expected.But L.monocytogenes strains did not express proper antigenic protein. CONCLUSIONS: Three novel L.ivanovii-based tuberculosis vaccine candicates,carrying Mycobacterium tuberculosis Rv0129c antigen gene cassette (coding for Ag85C) or Rv3875 gene cassette (coding for ESAT-6),and expressing relevant antigenic proteins have been successfully selected.

Nutrients recycling and biomass production from Chlorella pyrenoidosa culture using anaerobic food processing wastewater in a pilot-scale tubular photobioreactor.

Microalgae cultivation in anaerobic food wastewater was a feasible way for high biomass production and nutrients recycling. In this study, Chlorella pyrenoidosa culture on anaerobic food wastewater was processed outdoors using a pilot-scale tubular photobioreactor. The microalgae showed rapid growth in different seasons, achieving high biomass production of 1.83-2.10 g L-1 and specific growth rate of 0.73-1.59 d-1. The biological contamination and dissolved oxygen were controlled at suitable levels for algal growth in the tubular photobioreactor. Lipids content in harvested biomass was 8.1-15.3% of dried weight, and the analysis in fatty acids revealed high quality with long carbon chain length and high saturation. Additionally, algal growth achieved effective pollutants purification from wastewater, removing 42.3-53.8% of CODCr, 82.6-88.7% of TN and 59.7-67.6% of TP. This study gave a successful application for scaled-up microalgae culture in anaerobic food processing wastewater for biodiesel production and wastewater purification.

Intranasal vaccination with Listeria ivanovii as vector of Mycobacterium tuberculosis antigens promotes specific lung-localized cellular and humoral immune responses.

We have previously demonstrated that a recombinant Listeria ivanovii (LI) strain expressing the ESAT-6 or Ag85C protein of Mycobacterium tuberculosis (Mtb) as a tuberculosis (TB) vaccine candidates induced antigen-specific cellular immune responses after intravenous immunization of mice. However, whether such recombinant strains could induce desired immune responses in the lung, where TB infection occurs, is not clear. In this paper, C57BL/6 J mice were intranasally vaccinated with attenuated LIΔactAplcB-Rv3875 (Δ refers to gene deletion in the bacterial genome) or LIΔactAplcB-Rv0129c, the two vaccine candidates that utilize LI as an antigen delivery vector. Bacterial load in the target organs, histological changes in the infected organs, the percentage of specific cytokine-secreting T cells in the lung and spleen, IgG levels in the serum and secretory IgA (SIgA) levles in bronchoalveolar lavage (BAL) fluid were determined at specific days post inoculation (dpi). The results showed that both strains were mainly confined to the lung and were eliminated at 10 dpi. The histological damage caused by the infection in the lung was slight and recovered by day 5. Intranasal vaccination of the mice twice at an interval of 4 weeks notably elicited TB antigen-specific CD4+ and CD8+ T cell responses in the lung and SIgA secretion in the pulmonary mucosa, and significantly enhanced the percentage of double-functional CD8+ T cells (IFN-γ+ TNF-α+ CD8+). To our knowledge, this is the first report regarding the used of LI vector vaccines to induce promising lung-localized cellular and humoral immune responses by intranasal vaccination. These data suggest that LI could be a novel and promising live vector to construct an intranasal vaccine against respiratory diseases.