Rapid construction of a mammalian cell surface display library of full-length human antibodies / 南方医科大学学报
Journal of Southern Medical University
;
(12): 308-312, 2011.
Article
Dans Chinois
| WPRIM
| ID: wpr-307944
ABSTRACT
<p><b>OBJECTIVE</b>To construct a mammalian cell surface display library of full-length human antibodies.</p><p><b>METHODS</b>The total RNA was isolated from human peripheral blood mononuclear cells (PBMCs), and the genes encoding the heavy chain variable regions and kappa light chains (VH and Cκ) of the antibodies were amplified by RT-PCR. The amplified VH and Cκ gene sequences were separately inserted into the vector pDGB-HC-TM. The ligation mixtures were transformed into competent E.coli DH5α cells to construct the antibody libraries, and the library sizes and diversity were analyzed. The library DNAs were transfected into CHO cells and the expression of the full-length human antibodies on the surface of CHO cells was analyzed by flow cytometry.</p><p><b>RESULTS</b>The heavy chain gene library constructed showed a diversity of 2.6 × 10(5), and the kappa light chain gene library had a diversity of 2.0 × 10(5). Sequence analysis of 10 clones randomly selected from the constructed heavy chain gene library and 10 from the light chain gene library showed that 8 heavy chain clones and all 10 light chain clones contained correct open reading frames. Flow cytometry demonstrated that all the 18 clones expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces was detected with the positive cells reaching 31.5.</p><p><b>CONCLUSIONS</b>A full-length human mammalian display antibody library with a combinatory diversity of 5.2 × 10(10) can be constructed in two weeks, which allows the display of full-length antibodies on mammalian cell surface.</p>
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Données de séquences moléculaires
/
Transfection
/
Expression des gènes
/
Banque de gènes
/
Séquence d'acides aminés
/
Cellules CHO
/
Clonage moléculaire
/
Chaines lourdes des immunoglobulines
/
Chaines légères des immunoglobulines
/
Cytométrie en flux
Limites du sujet:
Animaux
/
Humains
langue:
Chinois
Texte intégral:
Journal of Southern Medical University
Année:
2011
Type:
Article
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