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Abstract
Abstract Objective Targeting the protein of interest to a particular tissue to achieve high-level expression is an important strategy to increase expression efficiency. The use of the plant seed oil body as a bioreactor can not only increase the amount of target protein, but also reduce the cost of downstream processing. Methods VEGF165 was expressed in Arabidopsis thaliana seeds via oilbody fusion technology. The pKO-VEGF165 vector was construted and transformed into A. thaliana seeds. T3 transgenic seeds was detected by SDS-PAGE and western blot methods. The cell activity was tested by MTT methods. Result The phaseolin promoter was used to drive seed-specific expression of the VEGF 165 gene in transgenic A. thaliana. The coding region of VEGF 165 was fused to the Arabidopsis oleosin sequence to target the protein to the oil bodies in the seeds of transgenic plants. The T-DNA region of recombinant plasmid pKO-VEGF165 was shifted to A. thaliana seeds via the floral-dip method. Protein was analyzed by electrophoresis and protein hybridization analyses. Finally, MTT assays showed that the oleosin-VEGF165 fusion protein played a part in the proliferation of HUVEC cells in vitro. Conclusion Oleosin-VEGF165 was successfully expressed and it had stimulated HUVEC cell proliferation activity.
Highlights
Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein that induces the production of mitogen in endothelial cells [1]
A variety of VEGF isoforms are possible to carry out different effects and they synergize in the tumor angiogenesis process
The literature reported that increased tumor angiogenesis and poor outcomes correlate with the overexpression of VEGF165 in colon cancer, osteosarcoma, renal cell carcinoma and non-small cell lung cancer [13,14,15]
Summary
Expression Oleosin-VEGF165 in Arabidopsis thaliana and screening for transgenic lines. The Oleosin-VEGF165 fusion protein was identified from T3 transgenic plant T3-1, T3-2, T3-3 by the anti-VEGF165 polyclonal antibody (Figure 3B). The. VEGF165 gene was expressed in A. thaliana and its product accumulated in the oil bodies in the transgenic seeds. Four independent transgenic lines was evaluated by extraction of the oil bodies which expressed oleosin-VEGF165 protein (Figure 3A and B). According to western blot result, transgenic line T3-1, T3-2, T3-3 were chosen to evaluate the bioactivity of the oleosin-VEGF165 fusion proteins. Because the oil bodies expressed oleosin-VEGF165 was obtained from T3-1, T3-2, T3-3 seeds, these seeds were used to detect the proliferative activity by the MTT method with HUVEC cells. The results showed that the oil bodies that expressed oleosin-VEGF165 fusion protein from the T3-1, T3-2, T3-3 had a dose-dependent proliferative effect on HUVEC cells. The wild-type oil bodies used as the negative control did not obviously show this dose-response relationship with cell proliferation
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