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Updated: Jun 5, 2026
Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
[Enhancing effect of AR-3 on protein splicing-based eukaryotic cell gene transfer of full-length coagulation factor
Fu-Xiang Zhu1, Ze-Long Liu, Jing Miao
1Biomedical Institute, Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
Objective:
To study the effect of an acidic region-3 (AR-3), capable of improving the secretion of heavy chain of coagulation factor VIII (fVIII), on the secretion of protein spicing ligated full-length fVIII.
Methods:
A pair of vectors was used to deliver intein fused heavy and light chain genes of a full-length fVIII gene with an additional AR-3 incorporated on the end of heavy chain into cultured 293 cells. The intracellular protein splicing was observed by Western blot. And the secretion of spliced fVIII and activity in culture supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) and Coatest assay respectively.
Results:
A noticeable spliced fVIII protein band was observed from the gene co-transfected cells. The culture supernatant displayed a spliced fVIII of (112 ± 18) ng/ml with an activity of (0.76 ± 0.13) U/ml greater than that of cells co-transfected with AR-3-free heavy chain and light chain genes [(64 ± 11) ng/ml and (0.37 ± 0.05) U/ml]. And a spliced fVIII of (27 ± 7) ng/ml with an activity of (0.16 ± 0.05) U/ml was detected in the culture supernatant from combined cells separately transfected with AR-3-fused heavy chain gene and light gene.
Conclusion:
AR-3 can enhance the fVIII gene transfer by improving the secretion of intein spliced full-length fVIII.
