Camel Heavy Chain Polyclonal Antibody Raised Against Recombinant Murine Placental Growth Factor Expressed in Escherichia coli.

Abstract

Angiogenesis, a neovascularization process, is the most important occurrence in developmental and pathological conditions. Key factors involve belonging to the vascular endothelial growth factor family. Recently, placental growth factor (PlGF) has been considered in medicine because of its pathological importance in solid tumor invasion and metastasis. Therefore, PlGF targeting plays a promising role in the hindrance of tumor progress. In this study, murine PlGF was cloned, expressed in an Escherichia coli system, and used for development of polyclonal camel antibody. Codon-optimized mouse PlGF (mPlGF) cDNA was synthesized and cloned in to pET-28a. The expression was performed in BL21 (DE3) E. coli strain and purified by immobilized metal affinity column chromatography. A camel was subcutaneously immunized six times over a one-week interval using purified protein. IgGs were purified by applying the serum on two sequential column affinity chromatography using proteins A and G. Then, anti-PlGF IgG was identified by Western bolt analysis and ELISA using commercial and expressed PlGF. Synthesized mPlGF was cloned successfully into the pET-28a expression vector, and the accuracy of construct was confirmed by map restriction analysis and sequencing. The expression was induced by 1 mM IPTG and confirmed by SDS-PAGE and Western blot using anti-His monoclonal antibody. The camel was immunized using recombinant mPlGF, and IgG was purified by two-step affinity chromatography. Identification of specific IgG against mPlGF was confirmed by ELISA assay.