Objective To explore the effect of exogenous adrenomedullin(ADM) on expressions of glutathion in plasma and brain tissue inneonatal rats with hypoxic-ischemia reperfusion brain damage(HIRBD) and the mechanism of action.Methods Fifty-six cases of 7 d SD rats were randomly divided into 4 groups including normal control group(without any treatment),HIRBD group(the model with hypoxia for 2 h and ischemia for 1 h),primed group(abdomen infusion of ADM at 0.5 h before making model,the other was same to the HIRBD group)and treatment group(abdomen infusion of ADM at onces after making model,the other was same to the HIRBD group).The neonatal rats in 4 groups were derived blood and brain tissue after decapitation at either 4 h or 24 h after reperfusion.The levels of gtutathion(GSH) in plasma and brain tissue were determined by using chromatometry.Results In HIRBD group,the affection areas at 24 h after reperfusion enlarged compared with those at 4 h after reperfusion.Meanwhile,the affection of punctiform degeneration or necrosis at 4 h after reperfusion transformed into the affection of lamellar or diffuse degeneration or necrosis at 24 h after reperfusion.The levels of GSH in plasma and brain tissue in HIRBD group at either 4 h or 24 h after reperfusion were significantly lower than that in normal control group(Pa0.05).Meanwhile the pathology score of brain section in primed group and treatment group were significantly lower than that in HIRBD group at either 4 h or 24 h after reperfusion(Pa0.05).Conclusion Exogenous ADM can induce the neuroprotection in HIRBD by adjusting the expression of GSH.

OBJECTIVELcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study.

METHODSA new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography.

RESULTSRemoval of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)₃ adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10⁶ CFU of Y. pestis virulent strain 141.

CONCLUSIONThe completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Amino Acid Sequence ; Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; immunology ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Female ; Genetic Vectors ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plague ; immunology ; prevention & control ; Plague Vaccine ; genetics ; immunology ; Plasmids ; Pore Forming Cytotoxic Proteins ; genetics ; immunology ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; genetics ; immunology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Survival Analysis ; Vaccines, Subunit ; genetics ; immunology ; Yersinia pestis ; growth & development ; immunology

OBJECTIVETo evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study.

METHODSGroups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization.

RESULTSThe immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively.

CONCLUSIONBALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.

Animals ; Antibodies, Bacterial ; blood ; Dose-Response Relationship, Immunologic ; Female ; Guinea Pigs ; Immunization ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plague ; prevention & control ; Plague Vaccine ; immunology ; Rabbits ; Vaccines, Subunit ; immunology