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. 2014 Jan;21(1):35-9.
doi: 10.1016/j.sjbs.2013.04.008. Epub 2013 May 7.

Recombinant expression and purification of human placental growth factor 1 and specific camel heavy chain polyclonal antibody preparation

Roghaye Arezumand  1 , Reza Mahdian  1 , Mahdi Behdani  2 , Hossein Khanahmad  3 , Jahangir Langari  1 , Nabiollah Namvarasl  4 , Reza Hassanzadeh-Ghasabeh  5 , Sirous Zeinali  1
Affiliations

Recombinant expression and purification of human placental growth factor 1 and specific camel heavy chain polyclonal antibody preparation

Roghaye Arezumand et al. Saudi J Biol Sci. 2014 Jan.

Abstract

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Unlike VEGF, PlGF is dispensable for normal cell development as well as playing various roles in pathological angiogenesis which occurs in tissue ischemia, inflammation, and malignancy. The PlGF-1 has been considered as a potential candidate for the diagnosis and targeting of pathological angiogenesis. Camelidae serum contains an important fraction of functional antibodies, called heavy-chain antibodies (HcAbs) that are naturally devoid of light chains. Camelid HcAbs recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody. Here, we describe the expression and purification of recombinant human PlGF-1 (rhPlGF-1). This protein was subsequently used for the preparation of camel heavy chain polyclonal antibody against rhPlGF-1. The recombinant expression plasmid pET-26b-hPlGF-1 was introduced into Escherichia coli BL21 cells to express the rhPlGF-1 protein. Purified rhPlGF-1 was used to immunize camel, the specific reactivity of HcAb was determined with ELISA and western blot. Western blot analysis indicated that the antiserum specifically reacted to the recombinant protein. The rhPlGF-1 protein and its antibody may be used for the development of detection assays needed for clinical research.

Keywords: Angiogenesis; Heavy chain antibody; PlGF; Polyclonal antibody; VHH.

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Figures

Figure 1
Figure 1
(a) Schematic expression cassette of pET26-hPLGF-1, (b) The amino acid sequence of hPLGF-1.
Figure 2
Figure 2
Gel filtration chromatography profile of the Ni–NTA purified rhPLGF-1, loaded onto a superdex 75 column in PBS buffer.
Figure 3
Figure 3
Expression and characterization of the rhPlGF-1. (a) SDS–PAGE. Lane1, before induction; Lane2, Induced with 1 mM IPTG; Lane3, Purified protein. (b) Western Blotting. Lane 1, purified rhPlGF-1 protein; Lane 2, commercial human PlGF-1. Lane M: protein molecular weight marker.
Figure 4
Figure 4
The SDS–PAGE pattern of purified camel antibodies in reduced condition. Lane 1, the conventional camel antibody (IgG1) with light and heavy chain. Lane 2, 3, the heavy chain antibodies (IgG2, 3 respectively).
Figure 5
Figure 5
Sensitivity analysis of HcAbs by ELISA. The camelid HcAbs antibody at different dilutions (1:200 to 1:50,000) reacted with the recombinant rhPLGF protein.
Figure 6
Figure 6
Determination of specificity by western blot analysis in non-reducing condition with Heavy-chain polyclonal antibody. Lane 1, purified rhPlGF-1, Lane 2, commercial human PlGF-1, M, protein molecular weight marker.
None
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