Abstract
Integrins are heterodimeric membrane proteins, which are exposed to post translational modifications in eukaryotic cells in contrast to prokaryotic cells. These modifications provide advantages for production of proper nanobody, mono and polyclonal antibody against this surface protein and also in aptamer selection process. Since the majority of diagnostic and therapeutic antibodies, target the surface epitopes, eukaryotic membrane proteins provide an appropriate model for further investigation on therapeutic agents. Escherichia coli strain top 10, was used as host for ITGA-4 expression vector encoding the human integrin α4. The plasmid was extracted and consequently, ITGA-4 vector was digested to make a linear plasmid. Human Embryonic Kidney-293 (HEK-293) cell transfected with linear plasmid and subsequently screened for stable ITGA-4 expressing Cells. Three separated clones were isolated twenty one days after transfection. Chromosomal DNA was extracted from ITGA-4-transfected cells. The presence of ITGA-4 gene in HEK-293 genome was confirmed by PCR. The expression level of ITGA-4 on HEK-293 cells was also analyzed by Flow cytometry. Flow cytometric analysis showed that HEK-293 cells have no expression of integrin α4 on their surface while 95% of transfected HEK-293 cells with ITGA4, expressed different levels of integrin α4 on their surfaces which correlates well with genomic DNA PCR amplification results. The results suggest that we have successfully constructed the integrin α4 expressing HEK293 cell, which will facilitate further research into the production of antibody, nanobody and aptamer against α4 integrin.
Highlights
Integrins are heterodimeric membrane proteins, which play a crucial role in cell adhesion and signal transduction.[1]
Flow cytometric analysis showed that Human Embryonic Kidney-293 (HEK-293) cells have no expression of integrin α4 on their surface while 95% of transfected HEK-293 cells with ITGA4, expressed different levels of integrin α4 on their surfaces which correlates well with genomic DNA Polymerase Chain Reaction (PCR) amplification results
The results suggest that we have successfully constructed the integrin α4 expressing HEK293 cell, which will facilitate further research into the production of antibody, nanobody and aptamer against α4 integrin
Summary
Integrins are heterodimeric membrane proteins, which play a crucial role in cell adhesion and signal transduction.[1]. The selected aptamers will be useful for detection and blockade of specific surface markers.[18] In 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 alpha[4] integrin that could inhibit alpha[4] 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
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