Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.
Biomed Environ Sci
; 23(5): 333-40, 2010 Oct.
Article
em En
| MEDLINE
| ID: mdl-21112480
ABSTRACT
OBJECTIVE:
LcrV 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.METHODS:
A 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.RESULTS:
Removal 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.CONCLUSION:
The 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peste
/
Yersinia pestis
/
Vacina contra a Peste
/
Engenharia de Proteínas
/
Proteínas Citotóxicas Formadoras de Poros
/
Antígenos de Bactérias
Limite:
Animals
Idioma:
En
Revista:
Biomed Environ Sci
Assunto da revista:
SAUDE AMBIENTAL
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
China