Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Plasmids, MultiBacmid, Reagents, and Genes
2.2. Construction of Transient Expression Plasmids and Transfection into Sf9 Cells
2.3. Extraction and Reverse Transcription of RNA and Quantitative PCR
2.4. Construction of Recombinant Plasmids for Early and Late Stable Expression of VSRs
2.5. Construction of Recombinant MultiBac and Generation of Recombinant Baculovirus
2.6. The Expression of Target Genes Was Determined Using Western Blotting
2.7. Flow Cytometry to Determine Apoptosis in Insect Sf9 Cells
2.8. Evaluation of the Recombinant Baculovirus Titers Using TCID50
2.9. Statistical Analysis
3. Results
3.1. The Normal Cellular Functions Are Maintained in Insect Cells That Overexpress VSRs
3.2. The Presence of VSRs Markedly Attenuated the Impact of Intracellular RNA Interference
3.3. The Presence of VSR Significantly Enhanced the Expression Efficiency of the Recombinant Protein in the MultiBac System
3.4. The Presence of VSRs Enhance the Production of Recombinant Viruses with Higher Titers in the MultiBac System
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Name | Sequence | Genes |
---|---|---|
p0 (with His-tag) | F: 5′-cccgggatgcatcatcaccaccatcacatgattgtattgacccagt R: 5′-gcatgcttattcttgtaattccttt | Potato leafroll virus p0 Gene |
p19 (with His-tag) | F: 5′-cccgggatgcatcatcaccaccatcacatggaacgagctatacaag R: 5′-gcatgcttactcgctttctttttcg | Tomato bushy stunt virus p19 Gene |
core (with His-tag) | F: 5′-cccgggatgcatcatcaccaccatcacatgagcacgaatcctaaac R: 5′-gcatgcttaagccgaggcgggaacg | Hepacivirus C core Gene |
Fluc | F: 5′-ggatccatggaagacgccaaaaacata R: 5′-tctagattacacggcgatctttccg | Firefly-luciferase Gene |
Rluc | F: 5′-cccgggatgacttcgaaagtttatgat R: 5′-gcatgcttattgttcatttttgagaact | Renilla-luciferase Gene |
egfp | F: 5′-ggatccatggtgagcaagggcgagga R: 5′-tctagattacttgtacagctcgtccat | Enhanced Green Fluorescent Protein Gene |
mCherry | F: 5′-cccgggatggtgagcaagggcgagga R: 5′-gctagcttacttgtacagctcgtccatgcc | mCherry Fluorescent Protein Gene |
Name | Sequence | Genes |
---|---|---|
α-Tubulin | F: 5′-agtccagatcggtaatgc R: 5′-gctgaagaaggtgttgaag | Reference Gene |
bvg-1 | F: 5′-cccgtaacggacctcgtactt R: 5′-ttatcgagatttatttgcatacaac | Baculovirus Genome |
Caspase-1 | F: 5′-gattcaaagttacggtgttcccta R: 5′-ggttgtctggcttgtaatgagtat | Cysteine Aspartate-specific Gene |
Caspase-2 | F: 5′-gtaaggttctgattggcaattagc R: 5′-cggtacttgtggttggtgtt | Cysteine Aspartate-specific Gene |
Caspase-9 | F: 5′-acacagagtttgacaacaatatcg R: 5′-ggtctcatagtccaccaacac | Cysteine Aspartate-specific Gene |
Dronc | F: 5′-ctggtagatacgcttggagaacta R: 5′-gcctgtttgatgtgctaagact | Apoptosis Gene |
Dredd | F: 5′-aacaccacaaggaatggaagt R: 5′-agttacaggcatcgttggaa | Apoptosis Gene |
Sf9-iap | F: 5′-gttggagagttgtgttgtttgttt R: 5′-aatagcgttaatgttgaggaggag | Inhibitor of Apoptosis Gene |
vp39 | F: 5′-acccgataagaagcagtgac R: 5′-cccagagtagcgttaggatt | Baculovirus Capsid Gene |
p35 | F: 5′-cgaacgcaacgactactac R: 5′-tgagcaaacggcacaataac | Apoptosis Gene |
p53 | F: 5′-caccgtctcaaccgtatc R: 5′-gaggacattcttcgctattt | Apoptosis Gene |
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Zheng, H.; Zhao, H.; Xiong, H.; Awais, M.M.; Zeng, S.; Sun, J. Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells. Insects 2024, 15, 375. https://doi.org/10.3390/insects15060375
Zheng H, Zhao H, Xiong H, Awais MM, Zeng S, Sun J. Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells. Insects. 2024; 15(6):375. https://doi.org/10.3390/insects15060375
Chicago/Turabian StyleZheng, Hao, Hengfeng Zhao, Haifan Xiong, Mian Muhammad Awais, Songrong Zeng, and Jingchen Sun. 2024. "Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells" Insects 15, no. 6: 375. https://doi.org/10.3390/insects15060375