In Vitro Evaluation of Vegf-Pseudomonas Exotoxin: A Conjugated on Tumor Cells.

BACKGROUND: Angiogenesis which occurs mandatory in solid tumors, is a critical step in malignancy progression. Vascular endothelial growth factor (VEGF) is mainly responsible for angiogenesis process and facilitates the formation of new vessels. Distribution of monoclonal antibodies against VEGF or VEGF receptor (VEGFR) into the solid tumors is limited because of their huge dimensions. Moreover, many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. MATERIALS AND METHODS: We designed, expressed and evaluated the cytotoxicity of a novel nano-immunotoxin composed of VEGF splice variant containing 121 amino acids (VEGF121) and truncated the exotoxin A of Pseudomonas aeruginosa (PE38-KDEL). The fusion protein VEGF121-PE38 was successfully cloned and expressed in Escherichia coli, purified by Ni+ 2 affinity chromatography. The fusion protein was subsequently subjected to refolding using the reduced and oxidized glutathione. RESULTS: The expression level of the fusion protein reached to 1 mg/ml. The VEGF121-PE38 immunotoxin showed a 59 KDa MW which had cytotoxic effect on HUVEC and 293/KDR cells as low and high expressing VEGFR2 cells, respectively. But the cytotoxicity on 293/KDR was 100 folds more than that of VEGFR2 low expressing cell HUVEC. CONCLUSION: The designed immunotoxin showed more selectivity for higher VEGFR2 expressing cells in vitro.

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

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.

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

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.