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.

[Molecular mechanism of ORFV intervention strategies based on the UPS of host cell: a review].

In order to compete the antiviral effects of the host cell in the process of infection, ORFV(known as Orf virus) relies on a series of functional genes developed through long-term population evolution, such as interferon resistance genes, Bcl-2 protein genes and cell cycle inhibitor gene and so on, with these weapons this virus is able to effectively counteract immune clearance and immune regulation from a host cell. Concurrently, ORFV also focuses on exploiting signal transduction pathways of the ubiquitin-proteasome system(UPS), circumvents the intracellular signal transduction and CD8+ T activation, for shielding virus particles towards maturation and releasing outside. This review introduced inner link between the UPS of host cell and intervention mechanism by virus, and analyzed the key roles of certains components in UPS, these all together showed the evolution tendency of ORFV that was involved in the designing of inhibition to immune response and for intracellular immune escape upon the selection pressure in host cell infected.

[Immunogenicity analysis of recombinant GST protein of Fasciola hepatica in SD rats].

OBJECTIVE: To analyze the immunogenicity of recombinant glutathione S-transferase protein of Fasciola hepatica (FhGST) in SD rats. METHODS: The recombinant expression plasmid pET30a-FhGST was transformed into E. coli BL21 (DE3) cells and induced with IPTG for protein expression. The recombinant protein FhGST was analyzed by SDS-PAGE and identified by Western blotting. Twenty SD rats were randomly divided into two groups: immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhGST protein. The adjuvant control group with 10 SD rats received only adjuvants emulsified with PBS. All the rats received three immunizations at 3-week intervals. Serum samples were collected at pre-immunization, the day after each immunization, 3 weeks and 6 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by MTT. RESULTS: The molecular weight of purified FhGST was about M(r) 31 300. The recombinant FhGST was recognized by pool sera of goats naturally infected with F. hepatica. The recombinant protein induced specific antibody IgG against GST protein in SD rats significantly higher than that of the control, and the antibody titer reached the peak at 9 weeks after the first immunization (GMT 1:89 144). FhGST protein significantly enhanced the growth and proliferation of rat splenocytes. CONCLUSION: The recombinant FhGST protein induces specific immune response in SD rats.