[Expression of recombinant human ZP3 protein using the baculovirus expression system].
Zhonghua Nan Ke Xue
; 20(11): 978-83, 2014 Nov.
Article
en Zh
| MEDLINE
| ID: mdl-25577831
ABSTRACT
OBJECTIVE:
To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3.METHODS:
The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored.RESULTS:
The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm.CONCLUSION:
It is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Glicoproteínas de Membrana
/
Transfección
/
Proteínas del Huevo
/
Baculoviridae
/
Receptores de Superficie Celular
Tipo de estudio:
Health_technology_assessment
Límite:
Humans
Idioma:
Zh
Revista:
Zhonghua Nan Ke Xue
Asunto de la revista:
MEDICINA REPRODUTIVA
Año:
2014
Tipo del documento:
Article
País de afiliación:
China