Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α.
Mol Med Rep
; 16(2): 1021-1030, 2017 Aug.
Article
en En
| MEDLINE
| ID: mdl-28586021
ABSTRACT
The aim of the present study was to construct a fast-acting, eukaryotic expression vector in eukaryotic cells based on transmembrane-tumor necrosis factorα (TMTNFα) structure. Two types of recombinant eukaryotic expression vectors were constructed, pcDNA3.1-TM-enterokinase-TNFα and pcDNA3.1TMFactor XaTNFα, according to the TNFα transmembrane segments. Following the generation of these vectors, mouse embryonic 3T3 fibroblasts were transfected and reverse transcriptionpolymerase chain reaction and western blotting analyses were used to analyze mTNFα mRNA and protein expression levels, respectively, in total cellular protein extracts and extracellular fluid. The biological activity of TNF-α in the extracellular fluid was then measured using an MTT assay. The vectors were successfully constructed, and mRNA and fusion proteins were detected in the 3T3 cells. Among the fusion proteins, the one observed in pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells remained as a transmembrane protein. In addition, treatment of L929 cells with TNFα derived extracellular fluid samples from pcDNA3.1TMFactorXaTNFαtransfected 3T3 cells was associated with a dosedependent reduction in in cellspecific activity. The results indicate that proteins expressed using pcDNA3.1TMFactorXaTNFα vectors form transmembrane proteins. In addition, the results indicate that, only when coupled with FactorXa activity, the extracellular region of TMTNFα forms sTNFα, and the controlled expression of the fusion protein is initiated.
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1
Banco de datos:
MEDLINE
Asunto principal:
Membrana Celular
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Factor de Necrosis Tumoral alfa
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Células Eucariotas
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Vectores Genéticos
Límite:
Animals
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Humans
Idioma:
En
Año:
2017
Tipo del documento:
Article