Characteristic Analysis of Ictalurus punctatus STING and Screening Validation of Interacting Proteins with Ictalurid herpesvirus 1
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell and Virus
2.2. RNA Extraction and cDNA Synthesis
2.3. Gene Cloning and Plasmid Construction
2.4. Sequence Analysis of IpSTING
2.5. Yeast Reporter Assay
2.6. Transient Transfection Assay
2.7. Co-IP Analysis and Western Blot
3. Result
3.1. Sequences Analysis of IpSTING
3.2. Autoactivation and Toxicity Tests of pGBKT7-ORF Bait
3.3. The ORF41 and ORF65 of CCV Potentially Interact with the IpSTING
3.4. Identification of the Interaction Between ORF41 or ORF65 and IpSTING by Co-IP
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer | Sequence (5′–3′) |
---|---|
AD-IpSTING-F | gtaccagattacgctcatatgATGGCGGAGGAGTGTGTGG |
AD-IpSTING-R | acgattcatctgcagctcgagTTATCTGTGTCTGTTGTGGTTATAGTGG |
BD-ORF1-F | tcagaggaggacctgcatatgATGGACGGTCTGAAGGAAATCA |
BD-ORF1-R | ttcggcctccatggccatatgTCAGGATAGCAGAAGGGTTTTTG |
BD-ORF2-F | tcagaggaggacctgcatatgATGACAGGTAGGCACTCGCCG |
BD-ORF2-R | ttcggcctccatggccatatgTCAGAGAGGTAAGAGTGGAGCATG |
BD-ORF3-F | tcagaggaggacctgcatatgATGGCATTCTCCACGTCTGACG |
BD-ORF3-R | ttcggcctccatggccatatgCTAGTTGAGGAGCGCACGTCG |
BD-ORF4-F | tcagaggaggacctgcatatgATGGCGTCCATCGATGTGG |
BD-ORF4-R | ttcggcctccatggccatatgTCAGAGCCAGTCCTGTCGATCG |
BD-ORF5-F | tcagaggaggacctgcatatgATGGCGCTCAGGGAAGGT |
BD-ORF5-R | ttcggcctccatggccatatgTTATAACTGACTTTTTCGTGTTAGGGG |
BD-ORF6-F | tcagaggaggacctgcatatgATGAACTCTCTCACTATCATCTTCCTCC |
BD-ORF6-R | ttcggcctccatggccatatgTCAGACCCGGATCTCCGC |
BD-ORF7-F | tcagaggaggacctgcatatgATGGCCGCCGTCATATTAGA |
BD-ORF7-R | ttcggcctccatggccatatgTCACAGGGAGTAATCGATGCG |
BD-ORF8-F | tcagaggaggacctgcatatgATGAAATGTCCGCGCGCA |
BD-ORF8-R | ttcggcctccatggccatatgTTATAGACCGGGGCTCCAGTC |
BD-ORF9-F | tcagaggaggacctgcatatgATGGCAACCAGACCCAAGG |
BD-ORF9-R | ttcggcctccatggccatatgTCACGGCGCGCAGGTGCA |
BD-ORF10-F | tcagaggaggacctgcatatgATGACCGTCAAGGGTTGCC |
BD-ORF10-R | ttcggcctccatggccatatgTTAGATGTACGGAGACATCATTGTCG |
BD-ORF11-F | tcagaggaggacctgcatatgATGAGGTGTATTCGAGCAGCG |
BD-ORF11-R | ttcggcctccatggccatatgTCAGTGGCCTGTTGGTGGAT |
BD-ORF12-F | tcagaggaggacctgcatatgATGACAAGCCCCCGCGAG |
BD-ORF12-R | ttcggcctccatggccatatgTCAGCGCGAACAGGCATC |
BD-ORF13-F | tcagaggaggacctgcatatgATGGCGTTTCTATTGCCCTTC |
BD-ORF13-R | ttcggcctccatggccatatgTCACCTGAGACCGTCCACGG |
BD-ORF14-F | tcagaggaggacctgcatatgATGGCGGAGAAGTTGATCCC |
BD-ORF14-R | ttcggcctccatggccatatgTTATATCTTGGAGAATTCGTCCACG |
BD-ORF15-F | tcagaggaggacctgcatatgATGGCAGCGGTTAATTGGC |
BD-ORF15-R | ttcggcctccatggccatatgTCAATGTTCGGGTTGGGTTG |
BD-ORF16-F | tcagaggaggacctgcatatgATGACATCTTCGGGATTCCTCA |
BD-ORF16-R | ttcggcctccatggccatatgTCACAATTTAAACTTTTCGATAAGCA |
BD-ORF17-F | tcagaggaggacctgcatatgATGATCCCGCCCGGGATC |
BD-ORF17-R | ttcggcctccatggccatatgCTACACGGTAGACCCGGGTTC |
BD-ORF18-F | tcagaggaggacctgcatatgATGAAAGCGTCCCAAGAGCG |
BD-ORF18-R | ttcggcctccatggccatatgTTATTCATCATCATCATCACCACCA |
BD-ORF19-F | tcagaggaggacctgcatatgATGGAAACAATGGGGCCCA |
BD-ORF19-R | ttcggcctccatggccatatgTTACTCGTTCGCGATAGTGTACG |
BD-ORF20-F | tcagaggaggacctgcatatgATGGCGTCCTCGGATAGGTT |
BD-ORF20-R | ttcggcctccatggccatatgTCACGATTCAGGATCAACAAGAAA |
BD-ORF21-F | tcagaggaggacctgcatatgATGTCTGCGACACAAGGTCCA |
BD-ORF21-R | ttcggcctccatggccatatgCTACTCGGACTCGGACTCGGA |
BD-ORF22-F | tcagaggaggacctgcatatgATGGGTCCCATACGGAAGCC |
BD-ORF22-R | ttcggcctccatggccatatgTTATTCAGAGTCTGAACCAGTGACACT |
BD-ORF23-F | tcagaggaggacctgcatatgATGTACATCCGTATTAATGAGTCCCG |
BD-ORF23-R | ttcggcctccatggccatatgTCACCAAAGGTTCCGAGGTTT |
BD-ORF24-F | tcagaggaggacctgcatatgATGACGTCCTCCATACCCGC |
BD-ORF24-R | ttcggcctccatggccatatgTCACTCGACGCACGCGGT |
BD-ORF25-F | tcagaggaggacctgcatatgATGGACGTTGAGATCAAAGAGTATGC |
BD-ORF25-R | ttcggcctccatggccatatgTCAATAAGGACATTCTCTCGGGA |
BD-ORF26-F | tcagaggaggacctgcatatgATGCAACTCGTTCGCGAAA |
BD-ORF26-R | ttcggcctccatggccatatgTCATATGATTTTAAGTCGACACTTGTTT |
BD-ORF27-F | tcagaggaggacctgcatatgATGGCGTCCATAACCGGTC |
BD-ORF27-R | ttcggcctccatggccatatgTCAAACACCTGAAAATTGTTTAATGA |
BD-ORF28-F | tcagaggaggacctgcatatgATGTCACTACCAGGCGGAAGAG |
BD-ORF28-R | ttcggcctccatggccatatgTTACAGATTCATGAGCGTCTCGA |
BD-ORF29-F | tcagaggaggacctgcatatgATGACCCGAAAAATTGACGC |
BD-ORF29-R | ttcggcctccatggccatatgTCAAATTCTTGACATGATACTGTCTCG |
BD-ORF30-F | tcagaggaggacctgcatatgATGATGGATATTTTCAATTCGGTG |
BD-ORF30-R | ttcggcctccatggccatatgTTACAACGGTGCAGCGGTACC |
BD-ORF31-F | tcagaggaggacctgcatatgATGTATTTACAAATGAATTTTTTTCCG |
BD-ORF31-R | ttcggcctccatggccatatgTTATTTCCCACTCGTGTATAAAATGAC |
BD-ORF32-F | tcagaggaggacctgcatatgATGAGGGGGGCGACCACG |
BD-ORF32-R | ttcggcctccatggccatatgTCACGAGACAATCGCTATCGAA |
BD-ORF33-F | tcagaggaggacctgcatatgATGGAGCCCATATCGTATGTCAC |
BD-ORF33-R | ttcggcctccatggccatatgTTACTCATCAGGAAGCTCGCCG |
BD-ORF34-F | tcagaggaggacctgcatatgATGGAACGGGTTTTGAGACAGT |
BD-ORF34-R | ttcggcctccatggccatatgTCAGTCTTGATCGCCGTGATC |
BD-ORF35-F | tcagaggaggacctgcatatgATGACCGCACCGATGTCTATAGA |
BD-ORF35-R | ttcggcctccatggccatatgTTATTCACAGAGGTCGAGGATCTTAT |
BD-ORF36-F | tcagaggaggacctgcatatgATGGACGGAATCTTCCCCAG |
BD-ORF36-R | ttcggcctccatggccatatgTCAAAAGGGCCATAGGAGCG |
BD-ORF37-F | tcagaggaggacctgcatatgATGTCAGGTGTTAAATTGGGGTCA |
BD-ORF37-R | ttcggcctccatggccatatgTCACACGTCTTCCATCGCCA |
BD-ORF38-F | tcagaggaggacctgcatatgATGTCGGTATTGGGAACGTGTT |
BD-ORF38-R | ttcggcctccatggccatatgTCAAGAATTTATATCATTGATGATACCTA |
BD-ORF39-F | tcagaggaggacctgcatatgATGGAAGCCATCGCACGTC |
BD-ORF39-R | ttcggcctccatggccatatgTCAGACGGCGTTCAGGTCG |
BD-ORF40-F | tcagaggaggacctgcatatgATGACACCAGTACCACCGAGTAGTC |
BD-ORF40-R | ttcggcctccatggccatatgTTACGTGCTTCCGGACGTG |
BD-ORF41-F | tcagaggaggacctgcatatgATGTATCTCAGGGATTTTCACGAGT |
BD-ORF41-R | ttcggcctccatggccatatgTTAAGGCGCACACACCCTG |
BD-ORF42-F | tcagaggaggacctgcatatgATGGCGTCCTTCGGAGAGC |
BD-ORF42-R | ttcggcctccatggccatatgTTACATTTCCAGGAAGAGGAACG |
BD-ORF43-F | tcagaggaggacctgcatatgATGGATGCACTCCGCAAGAA |
BD-ORF43-R | ttcggcctccatggccatatgTTAATCCTCATCATCACCTTCGATA |
BD-ORF44-F | tcagaggaggacctgcatatgATGGGCACCGCCGATGAG |
BD-ORF44-R | ttcggcctccatggccatatgTCATTCTGAAACTGGAAGGTCATTG |
BD-ORF45-F | tcagaggaggacctgcatatgATGACCTTCCAGTTTCAGAATGAGT |
BD-ORF45-R | ttcggcctccatggccatatgTTATTTATCAGAAAACGTTTTCTCAACA |
BD-ORF46-F | tcagaggaggacctgcatatgATGAAGAAAACGATGCTCGCA |
BD-ORF46-R | ttcggcctccatggccatatgTCAATACATCATCCCCATCGG |
BD-ORF47-F | tcagaggaggacctgcatatgATGATTGCGAGCATCGTTTTC |
BD-ORF47-R | ttcggcctccatggccatatgTCACCGGATCCCGTGACA |
BD-ORF48-F | tcagaggaggacctgcatatgATGAACAACGGGTGGGACG |
BD-ORF48-R | ttcggcctccatggccatatgTCACCAGATAATGGGTAGTTGATCA |
BD-ORF49-F | tcagaggaggacctgcatatgATGGGTGAGATGACCTCCGG |
BD-ORF49-R | ttcggcctccatggccatatgTCATTGTCCTGATGAACCAAACC |
BD-ORF50-F | tcagaggaggacctgcatatgATGGAATTACTTGTAGTGACCCTCAC |
BD-ORF50-R | ttcggcctccatggccatatgTTACCGCCTGGCTCCACTC |
BD-ORF51-F | tcagaggaggacctgcatatgATGGCCCAGTATATAGTGACGATATT |
BD-ORF51-R | ttcggcctccatggccatatgTCATACTCCCCAGATAGATAGTTGCA |
BD-ORF52-F | tcagaggaggacctgcatatgATGGAACAACAGAACCCGATACC |
BD-ORF52-R | ttcggcctccatggccatatgTCAAAAGGATACGACTTCTCGTTTAG |
BD-ORF53-F | tcagaggaggacctgcatatgATGCCCATGATGAACACCAAC |
BD-ORF53-R | ttcggcctccatggccatatgCTAAACGAGAAGTCGTATCCTTTTGA |
BD-ORF54-F | tcagaggaggacctgcatatgATGGGCATCAGGAAGAAGCTG |
BD-ORF54-R | ttcggcctccatggccatatgTCACTTTCGACCGAGCGTG |
BD-ORF55-F | tcagaggaggacctgcatatgATGTCTCTCACGAACAAACCCAG |
BD-ORF55-R | ttcggcctccatggccatatgTCAGGAATCGTGGAGGGCA |
BD-ORF56-F | tcagaggaggacctgcatatgATGTCGAGTGAAGTGTTCCGATG |
BD-ORF56-R | ttcggcctccatggccatatgTTATCGGTTCATCAATCTCTGAATTT |
BD-ORF59-F | tcagaggaggacctgcatatgATGGTCGGCAAAGGTCTCCCC |
BD-ORF59-R | ttcggcctccatggccatatgTCACGCCCGGGCAGGTG |
BD-ORF60-F | tcagaggaggacctgcatatgATGATGATTTTGTCAAAGGCCC |
BD-ORF60-R | ttcggcctccatggccatatgTCACGTGGAACTTTTGGGC |
BD-ORF61-F | tcagaggaggacctgcatatgATGATCTCCGTCGCGCGA |
BD-ORF61-R | ttcggcctccatggccatatgTCAGACGAGGGCCCAAAAGTT |
BD-ORF63-F | tcagaggaggacctgcatatgATGTACGTTGTCGTGTCGGTC |
BD-ORF63-R | ttcggcctccatggccatatgTCACTGTCCACAGAATCCGAC |
BD-ORF64-F | tcagaggaggacctgcatatgATGTCATCCCCGAGGGGG |
BD-ORF64-R | ttcggcctccatggccatatgTCAGCCAATAACTCCGGTGGATA |
BD-ORF65-F | tcagaggaggacctgcatatgATGGAAATAAACGCTGCAATC |
BD-ORF65-R | ttcggcctccatggccatatgCTATATGATTCGCGCTTAAAAACAAT |
BD-ORF66-F | tcagaggaggacctgcatatgATGGATTCCATCACGTTGA |
BD-ORF66-R | ttcggcctccatggccatatgTTATTTCCATTTATCCAGCAG |
BD-ORF67-F | tcagaggaggacctgcatatgATGGAATCCATTGTTTATGATAGTG |
BD-ORF67-R | ttcggcctccatggccatatgTCACTGCCCTTGAGGCGT |
BD-ORF68-F | tcagaggaggacctgcatatgATGTTGAGGACGATCGGTTCAC |
BD-ORF68-R | ttcggcctccatggccatatgTCATCCGATGTGGACGTTCAA |
BD-ORF70-F | tcagaggaggacctgcatatgATGGACGGTTACTTCAAAAATATC |
BD-ORF70-R | ttcggcctccatggccatatgTTACACACAAAAATGTTTAAACAGTAAC |
BD-ORF73-F | tcagaggaggacctgcatatgATGGCAGATAGGACACCCAAGC |
BD-ORF73-R | ttcggcctccatggccatatgTCAACTTAGTGTCCCGCTGGC |
BD-ORF74-F | tcagaggaggacctgcatatgATGAGCGGGAAGGTCGAAA |
BD-ORF74-R | ttcggcctccatggccatatgTCACCGACTGCCCGTGAT |
BD-ORF75-F | tcagaggaggacctgcatatgATGGTCAATACATTAAAAAGTTCCC |
BD-ORF75-R | ttcggcctccatggccatatgTCATGACAGCGACTCGAGG |
BD-ORF76-F | tcagaggaggacctgcatatgATGGACCGGCATCGGTAT |
BD-ORF76-R | ttcggcctccatggccatatgTCACGGGAATATTCTCGGG |
BD-ORF77-F | tcagaggaggacctgcatatgATGGCACGGAAAGGAGAGC |
BD-ORF77-R | ttcggcctccatggccatatgTCACGGTAAGAATGTCGACACC |
BD-ORF78-F | tcagaggaggacctgcatatgATGGAGTTATCACTGTACCGCG |
BD-ORF78-R | ttcggcctccatggccatatgTCAGTGTGAATTATTGGAATGGG |
BD-ORF79-F | tcagaggaggacctgcatatgATGGATTTCGAATGCGGTTC |
BD-ORF79-R | ttcggcctccatggccatatgTCAGCCGGTCTGGACGC |
pCDNA-ORF41-F | tagtccagtgtggtggaattcATGTATCTCAGGGATTTTCACGAGT |
pCDNA-ORF41-R | tgctggatatctgcagaattcAGGCGCACACACCCTGACT |
pCDNA-ORF65-F | tagtccagtgtggtggaattcATGGAAATAAACGCTGCAATCG |
pCDNA-ORF65-R | tgctggatatctgcagaattcTATATGATTCGCGCTTAAAAACACTG |
pCMV-Myc-STING-F | tccaagcttctgcaggaattcATGGCGGAGGAGTGTGTG |
pCMV-Myc-STING-R | tctgtcgacgatatcgaattcTCTGTGTCTGTTGTGGTTATAGTG |
Gene Name | Insert Type | Expression Vector | Application System | Cloning Method |
---|---|---|---|---|
ORF1–ORF73 | Viral coding regions | pGBKT7 | Y2HS, bait plasmids | One Step Cloning |
ORF41 | Viral coding regions | pcDNA3.1/His | Co-IP | One Step Cloning |
ORF56 | Viral coding regions | pcDNA3.1/His | Co-IP | One Step Cloning |
IpSTING | Host gene | pGADT7 | Y2HS, prey plasmid | One Step Cloning |
IpSTING | Host gene | pCMV-C-Myc | Co-IP | One Step Cloning |
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Meng, L.; Li, S.; Chen, H.; Yuan, S.; Zhao, Z. Characteristic Analysis of Ictalurus punctatus STING and Screening Validation of Interacting Proteins with Ictalurid herpesvirus 1. Microorganisms 2025, 13, 1780. https://doi.org/10.3390/microorganisms13081780
Meng L, Li S, Chen H, Yuan S, Zhao Z. Characteristic Analysis of Ictalurus punctatus STING and Screening Validation of Interacting Proteins with Ictalurid herpesvirus 1. Microorganisms. 2025; 13(8):1780. https://doi.org/10.3390/microorganisms13081780
Chicago/Turabian StyleMeng, Lihui, Shuxin Li, Hongxun Chen, Sheng Yuan, and Zhe Zhao. 2025. "Characteristic Analysis of Ictalurus punctatus STING and Screening Validation of Interacting Proteins with Ictalurid herpesvirus 1" Microorganisms 13, no. 8: 1780. https://doi.org/10.3390/microorganisms13081780
APA StyleMeng, L., Li, S., Chen, H., Yuan, S., & Zhao, Z. (2025). Characteristic Analysis of Ictalurus punctatus STING and Screening Validation of Interacting Proteins with Ictalurid herpesvirus 1. Microorganisms, 13(8), 1780. https://doi.org/10.3390/microorganisms13081780