Construction and Characterization of Recombinant Hek Cell over Expressing Α 4 Integrin

Introduction Integrins are heterodimeric membrane proteins, which play a crucial role in cell adhesion and signal transduction. 1 Integrins consist of an α and a β subunit in humans. 18 alpha and 8 beta subunits have been identified that form at least 24 different heterodimers through different combination of α and β subunits.


Introduction
Integrins are heterodimeric membrane proteins, which play a crucial role in cell adhesion and signal transduction. 1Integrins consist of an α and a β subunit in humans.18 alpha and 8 beta subunits have been identified that form at least 24 different heterodimers through different combination of α and β subunits. 2ntegrins mediate cell-cell and cell-Extra Cellular Matrix (ECM) adhesion, providing adhesion for adherent cells, drive many signalling pathways that regulate diverse processes including proliferation, migration, cell survival, differentiation, tumor invasion and metastasis. 3,4 4 β 1 is a receptor for the immunoglobulin adhesion ligand, Vascular Cell Adhesion Molecule 1 (VCAM-1), and fibronectin which are expressed on endothelial cell and ECM respectively.α 4 β 1 integrin is expressed at moderate-to-high levels on almost all lymphocytes, monocytes and eosinophils. 5[8] α 4 β 1 integrin-mediated cell-cell adhesions, plays a key role in tumor angiogenesis through homing of both endothelial progenitor cells and monocytes to neovascular tissue.Moreover α 4 β 1 integrin is essential for inflammation process, through leukocytes attachment to vascular endothelial cells during extravasation. 9,10inding of α 4 β 1 to VCAM-1, provides tumor angiogenesis and homing of hematopoietic stem and progenitor cells. 11nteraction of circulating leukocytes, with endothelium of the blood-brain barrier (BBB) and intestine is a critical step in pathogenesis of inflammatory diseases of the Central Nervous System (CNS) and Crohn's disease (CD).Previous studies, demonstrated T lymphocyte interaction, with the vascular endothelial cells of brain and intestine through α 4 integrin binding to VCAM-1 and mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) respectively. 12,13α 4 integrin-mediated trafficking of pathogenic effector T cells to the brain and intestine has been considered as a integrin involvement in tumor angiogenesis, it also could be authorised as a target for cancer therapy. 16ysabri (natalizumab), an antibody which blocks α 4 integrin and inhibits the α 4 -mediated adhesion of leukocytes to their counter receptors, has been used for treatment of patients with relapsing remitting multiple sclerosis (RRMS) and CD. 17 As monoclonal antibodies (mAbs) have high immunogenicity and molecular weight and low heat stability, are not in favour of treatment approaches.Thus research on other agents lacking the above disadvantages seems crucial for mAbs replacement.Aptamers are new tools with low immunogenicity and molecular weight and high heat stability which make them appropriate candidates for mAbs substitution.Aptamers are selected by a process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX).One of the most common SELEX methods is cell SELEX.In this method, those aptamers would be selected that bind to protein targets expressed on the cell surface with high specificity and affinity.Hence, type of cells used in this selection process is a critical parameter in cell SELEX.The selected aptamers will be useful for detection and blockade of specific surface markers. 18n the present study, we intended to construct an overexpressing full length of human ITGA-4 recombinant HEK cell (Hek293/ITGA-4).This construct could further be employed in SELEX process to select a specific aptamer or immunization of camel to generate nanobody against alpha4 integrin that could inhibit alpha4 integrin as a mediator of adhesion and migration.This aptamer could be utilized in preclinical studies for treatment of MS and Crohn's disease in future.

Transformation
Escherichia coli strain top 10 (Pasture, Tehran), was used as host for Z2827-M67-(ITGA4) expression vector (GeneCopoeia, MD, USA) encoding the human integrin α 4 .E.coli top 10 was grown in Luria-Bertani (LB) (Sigma-Aldrich, St. Louis, MO, USA) broth medium, containing tetracycline (50 µg/ml), under aerobic condition and shaking at 250 rpm at 37°C.E.coli top 10, was then transformed with Z2827-M67-(ITGA4) expression vector in order to plasmid amplification using the CaCl 2 procedure. 19The transformed cells were spread on LB agar plates containing 100 µg/ml ampicillin and incubated at 37°C overnight.Isolated single colonies were inoculated into LB broth with ampicillin (100 µg/ml) and grown at 37°C for 24 hours with shaking at 250 rpm for the purpose of plasmid extraction.The plasmids were extracted using commercial plasmid extraction kits (Feldan, Canada).Consequently, Z2827-M67-(ITGA4) vector was digested with Eco31I (Thermo Scientific, Waltham, MA, USA) enzyme according to manufacturer's instruction to make a linear plasmid.Digested plasmid was evaluated by agarose gel electrophoresis (Figure 1).

DNA extraction and Polymerase Chain Reaction (PCR) on genomic DNA
Chromosomal DNA was extracted from ITGA4transfected cells using commercial extraction kit (BIONEER, Korea) according to manufacturer's instructions.The presence of ITGA-4 gene in HEK-293 genome was assayed by PCR.Amplification of extracted genomic DNA was performed using specific primers designed for partial human ITGA-4 cDNA (forward ‫-׳5‬ cgggatccatgtggagctggaag-3‫׳‬ and reverse ‫-׳5‬ cgggatcctcagcggcgtttgagt-3‫.)׳‬All PCR reagents were purchased from Thermo Scientific (Waltham, MA, USA).The reactions performed using Biorad thermal cycler (Bio-Rad Laboratory, USA).PCR conditions were optimized as follows: a total reaction mix of 50 μl contained 250ng of genomic DNA as template, 100 pmol of each primer, 1 U of Taq DNA polymerase, 1.5mM MgCl2, 200 mmol of each dNTP, and the volume was adjusted to 50 μl with deionised water.PCR program was 94° C for 3 min and 30 cycles of 94° C for 30 sec, 58° C for 30 sec, and 72° C for 2 min and 30 sec followed by a final extension step of 72°C for 10 min.Gel electrophoresis was performed after PCR amplification on 1% agarose gel (Figure 2).
Flow cytometric analysis 100 μl of Hek293/ITGA-4 and HEK-293 cell suspension staining were done with 5 μl anti-human CD49d antibody (Biolegend, CA, USA) conjugated with phycoerythrin (PE) for 30 min at 4°C.Isotope control was used to enable correct compensation and to confirm antibody specificity.Flow cytometric analysis was performed on FACS Callibur Flow cytometer (BD bioscience, NJ, USA) by accumulating up to 10,000 events per tube.

Results and Discussion
Twenty one days after transfection, there were three separated clones in 100 mm plate.

Chromosomal DNA extraction and amplification
Genomic DNA from transfected cells was extracted.Primers were chosen to target the 900bp portion of the ITGA-4 cDNA.The PCR product illustrated amplification of a 900bp DNA band on agarose gel (Figure 2).

Flow cytometric analysis
As shown in Figure 3, HEK-293 cells have no expression of α 4 integrin on their surface while 95% of transfected HEK-293 with ITGA4 were expressed α 4 integrin which correlates well with genomic DNA PCR amplification results.Different eukaryotic proteins have been used in immunization of mouse and camel to produce nanobody, mono, and polyclonal antibody and in SELEX process to select specific aptamers.Eukaryotic proteins usually have post translational modifications e.g.glycosylation, phosphorylation and fatty acid addition.The majority of diagnostic and therapeutic antibodies, target the special epitopes which contain post translational modification residues.Eukaryotic proteins could be expressed in prokaryotic expression systems in high yield and low cost however; these proteins lack post translational modifications and even proper folding.On the other hand, although protein expression in eukaryotic expression systems like mammalian cell lines has low yield and the resulted proteins are expensive, the resulted protein has an accurate folding and appropriate post translational modifications.Displaying eukaryotic membrane or even secreting and intracellular proteins on surface of mammalian cell lines e.g.HEK, is an alternative method to overcome the above cited obstacles.The HEK-293 overexpressing ITGA-4 may provide an appropriate model for further investigation on diverse therapeutic agents.The existence of protein as target, on cell surface has advantages that stand it out comparing to other methods for biomolecule selection.Live cells with overexpression of a certain molecule, are required for production and selection process of biomolecules e.g.mono and polyclonal antibodies, nanobodies and aptamers.Most antibody targets are membrane proteins with post translation modifications, which are difficult to express and purify as recombinant proteins.The availability of such proteins in sufficient quantities for immunization and selection is a hurdle in all antibody technology platforms, thus using model cells overexpressing the desired protein could resolve this problem.The present model provides an approach for antibody production through minimizing impasses.A set of related molecules with antibodise are nanobodise founded in some animals e.g., camelides.Nanobodies are unique class of antibodies lacking L chains, thus, form a homodimeric H2-type (vHH) antibodies.In case of nanobody production, immunization of camel with peptides (even coupled to larger protein carriers) is not recommended as the majority of nanobodies distinguish conformational epitopes and a poor vHH response would rise subsequent to peptide injection.DNA immunization, cell immunization and DNA prime/cell boost are potential alternatives to protein immunization, thus presence of such a model seems crucial for nanobody production. 20nother group of high specificity biomolecules are aptamers.Aptamers have been compared to antibodies on the basis of their target-recognition capability, selective binding and high affinity, however aptamers have advantages over antibodies including low blood residence time, low toxicity or immunogenicity, long shelf life, high affinity and specificity, thermal stability, low cost, unlimited applications and chemically synthesis possibility. 21

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Over expression of α4 integrin for aptamer SELEX Advanced Pharmaceutical Bulletin, 2015, 5(3), 429-434 Aptamers usually are selected by screening nucleic acid libraries through SELEX process.Purified recombinant proteins are traditional targets for aptamer SELEX, although these proteins might have an impaired three dimensional structure and/ or post translational modification.Aptamers required native state of target molecule with their natural folding and modifications for a better isolation in SELEX process.The present HEK-293 cell over expressing α 4 integrin provides the native state of α 4 integrin with appropriate post translational modification on the cell surface, thus offers an appropriate tool for specific aptamer selection against α 4 integrin. 22

Conclusion
In this paper, we constructed a recombinant cell line (Hek293/ITGA4) over-expressing α 4 integrin.The expression of α 4 chain on cell surface was characterized by flow-cytometric staining.The results indicated that the ITGA-4 expression vector was successfully transfected into target cell, HEK-293.A highly and stably expressed α 4 integrin cell line obtained with hygromycin B antibiotic selection which could be applied in future investigations.