Effect of P19 gene expression on transcription rate and production of recombinant human tissue plasminogen (rtPA) in tobacco (nicotiana benthamiana)

Document Type : Research Paper

Authors

1 Department of Genetics and Plant Breeding, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

2 Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

Abstract

Objective
Tissue plasminogen activator is one of the most important drugs in the treatment of heart disease. This drug is produced in the expression system as a recombinant protein that has high production costs. Transient expression system is very suitable for protein expression because of its high expression, high speed, low cost and no spatial effect. Post-transcriptional silencing has been shown to affect expression levels. Therefore, the aim of this study was to investigate the effect of simultaneous expression of P19 silencing suppressor gene on transient expression of recombinant tissue plasminogen activator (rtPA) at transcriptional and protein levels in Nicotiana benthamiana.
 
 
Material and methods
To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pCAMBIA1304-rtPA with agrobacterium containing pCAMBIA1304-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pCAMBIA1304-rtPA. Leaf samples were prepared on 4, 7, and 10 day post-inoculation with Agrobacterium. Transcription and then protein levels were calculated using the Real Time PCR and ELISA tests.
 
Results
The results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 µg.g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively.
 
 Conclusion
The results showed that the use of transient expression method could be a suitable method for rtPA protein production.

Keywords

References

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Agricultural Biotechnology Journal
Volume 12, Issue 2
September 2020
Pages 103-128

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