Construction of a recombinant Neisseria gonorrhoeae strain with rmp gene deletion and study on the bactericidal activities of its antibodies / 中华微生物学和免疫学杂志

Objective To study the role of the outer membrane protein Rmp of Neisseria gonor-rhoeae strain in immunosuppression and the strategy of eliminating it .Methods The rmp gene of Neisseria gonorrhoeae strain was amplified by PCR and inserted into pMD 19-T vector .The recombinant vector pMD 19△rmp∷Kan containing Kan and the 5′-and 3′-flanking regions of rmp (△rmp∷Kan) was constructed by replacing 200 nucleotide residues of pMD 19-rmp with kanamycin resistance gene Kan and transformed into Neisseria gonorrhoeae WHO-A strain.PCR and Western blot assay were used to screen and identify the re-combinant mutant strains that could not express Rmp .Mice were immunized with mutant strains and bacteri-cidal activities of the immune sera were detected by antibody-mediated complement-dependent cytotoxicity assay.Results The mutant strains that could not encode Rmp were successfully constructed .Antibodies in-duced by mutant strains showed stronger bactericidal activity against Neisseria gonorrhoeae in comparison with those induced by wild strains .Conclusion The recombinant Neisseria gonorrhoeae strain with rmp gene de-letion might eliminate the immunosuppressive effects of Rmp expressed in wild gonococcal strains , which provides a reference for further development of novel live attenuated whole-cell vaccines of Neisseria gonor-rhoeae.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK) gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEÄ), thymidine kinase (BoHV-5TKÄ) and both proteins (BoHV-5gEÄTKÄ). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEÄ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKÄ (N = 8) or BoHV-5gEÄTKÄ (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKÄ and BoHV-5gEÄTKÄ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

Construction of GST/HIF-1α Fusion Protein Expression Vectors and the Expressions in Escherichia coil / 中国医科大学学报

Objective To construct GST/HIF1α fusion protein expression vector and induce its expression in Escherichia coli (E.coli). Methods The coding sequence of hypoxia inducible factor-la (HIF-la) and its deletion fragments were amplified from the plasmid pcD-NA3.1-HIF-la by PCR and inserted into pGEX-4T-2 by BamHI and Not I. The positive recombinanls were identified by restriction endonu-clease digestion and DNA sequencing. Then they were transformed into E.coli BL21, induced by IPTG and identified by SDS-PAGE and Western blot. Results The prokaryotic expression plasmid pGEX-4T-2-HIF-lα and its deletion mutants were successfully constructed and confirmed by enzyme digestion and sequencing. The desired CST/HIF-1α fusion proteins were expressed and confirmed by Western blot. Conclusion The prokaryotic expression plasmid of HIF-la and its deletion mutants were successfully constructed and the expression of fu-sion proteins was confirmed. This study provides the basis for the further research on purifying HIF-la protein and the biological function of HIF-lα.