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Cloning and Expression of the Functional Human Anti-vascular Endothelial Growth Factor (VEGF) Using the pcDNA3.1 Vector and the Human Chronic Myelogenous Leukemia Cell Line K562

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Abstract

In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies.

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Abbreviations

VEGF:

Vascular endothelial growth factor

HR:

Homologous recombination

CHO:

Chinese hamster ovary

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

HRP:

Horse radish peroxidase

bp:

Base pair

Hygro:

Hygromycin

Neo:

Neomycin

RPM:

Rotation per minute

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Authors and Affiliations

  1. Department of Molecular Genetics, Science and Research Branch, Islamic Azad University, Fars, Iran

    Masoumeh Hajirezaei & Mojtaba Darbouy

  2. Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Bahram Kazemi

  3. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Bahram Kazemi

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  1. Masoumeh Hajirezaei
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  2. Mojtaba Darbouy
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  3. Bahram Kazemi
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Correspondence to Bahram Kazemi.

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Hajirezaei, M., Darbouy, M. & Kazemi, B. Cloning and Expression of the Functional Human Anti-vascular Endothelial Growth Factor (VEGF) Using the pcDNA3.1 Vector and the Human Chronic Myelogenous Leukemia Cell Line K562. Protein J 33, 100–109 (2014). https://doi.org/10.1007/s10930-013-9533-y

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