Design and construction of Beclin1-expressing plasmid as an autophagy inducing system: a novel strategy for enhancing the potency of DNA vaccines

Abstract

Introduction: Autophagy is a complicated process which is involved in many biological events such as antigen presentation by immune cells. Beclin1, as a key component of autophagic machinery, plays the main role in the induction and initiation of this process. In the present study, we hypothesized that overexpression of Beclin1 could be useful in autophagy induction as an immunostimulatory strategy for improving the efficiency of DNA vaccines. Methods: Beclin1 gene was cloned into pVITRO2 eukaryotic expression vector and was confirmed by agarose gel electrophoresis. Beclin1 expression was evaluated by Real-Time RT-PCR and Western blotting. The autophagy induction by pVITRO2 encoding Beclin1 in HEK293 cells was evaluated through detection of the expression levels of LC3II as a marker for autophagy induction by Western blotting. Results: Beclin1 expression by the constructed plasmid was detected in HEK293 cells. The pVITRO2 encoding Beclin1 showed autophagy enhancement as compared to the control condition. Our data showed significant increase in the expression of LC3II protein following Beclin1 transfection (P ˂0.05 or 0.01). Conclusion: Our results support a novel strategy for autophagy induction via up-regulation of Beclin1 expression. This could be useful for improvement of DNA vaccines through enhancing their antigen presentation.

Antitumor Effects of HPV DNA Vaccine Adjuvanted with Beclin-1 as an Autophagy Inducer in a Mice Model

“…The construction, amplification, and purification of plasmid pcDNA3.1 expressing HPV-16 E7 have been described previously, and the expression of E7 from pcDNA3.1 was performed in CHO cells [ 22 , 23 ] . The construction, amplification, and purification of plasmid pVITRO2 expressing Beclin-1 have formerly been described, and expression of Beclin-1 from pVITRO2 was performed in HEK293 cells [ 24 ] . Briefly, the plasmids were generated by retrieving HPV-16 E7 and Beclin-1 sequences from the GenBank database and cloned into plasmids.…”

“…To evaluate the expression of genes from constructed plasmids, the proteins were separated by SDS polyacrylamide gel electrophoresis, blotted on a membrane and incubated with the specific monoclonal antibody. Details of each method and results are available elsewhere [ 22 - 24 ] .…”