[Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells].
The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.
Penteado
Flora Cristina Lobo
FC
Laboratório de Pesquisa, Centro Regional de Hemoterapia, Hospital das Clinicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. floralobo@pegasus.fmrp.usp.br
Medeiros
Luciene
L
Orellana
Maristela Delgado
MD
Palma
Patricia
P
Fontes
Aparecida Maria
AM
Takayanagui
Osvaldo Massaiti
OM
Covas
Dimas Tadeu
DT
D000818:Animals; D019070:Cell Lineage; D019976:Cloning, Organism; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D008322:Mammals / Q000821:virology*; D008562:Membrane Glycoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014759:Viral Envelope Proteins / Q000235:genetics*
D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins