Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity
Abstract
:1. Introduction
2. Results
2.1. MBOV_RS02825 Sequence Has a Typical Nuclease Structure
2.2. Expression of rMbovNase in Escherichia coli
2.3. Determinants of rMbovNase Nuclease Activity
2.4. rMbovNase Breakdown of NETs
2.5. Localization of MbovNase in M. bovis Cells
2.6. TNASE_3 Is Essential for rMbovNase Binding and Internalization in BoMac Cells
2.7. rMbovNase-Induced Cytotoxicity and Apoptosis of BoMac Cells
2.8. Expression of Some Signal Molecules Associated with Apoptosis
3. Discussion
3.1. rMbovNase Is a Membrane Bound and Secretory Nuclease
3.2. Binding, Internalization, and Cytotoxicity of rMbovNase
4. Materials and Methods
4.1. Ethics Statement
4.2. Bacterial Strains, Plasmids and DNA Manipulation
4.3. Cell Culture Conditions
4.4. Computer-Assisted Sequence Analysis
4.5. Cloning and Expression of MBOV_RS02825 and Generation of Antiserum
4.6. Analysis of rMbovNase Nuclease Activity
4.7. Zymogram Analysis
4.8. NETs Formation and Degradation Assay
4.9. Localization of rMbovNase by Western Blot Analysis
4.10. rMbovNase Binding and Invasion Assay
4.11. MTT Assay of Cell Viability after rMbovNase Treatment
4.12. Detection of Cell Apoptosis Induced by rMbovNase
4.13. Western Blot Assay of Molecular Expression Related to Apoptosis and the NF-κB Signal Pathway
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Names | Primer Sequences (5′-3′) | Positions |
---|---|---|
MBOV_RS02825 | ||
580F1 | CGGGGTACCGAAAATGGCACAATTAAG | 79→97 |
580R1 | AGTTTTTTCTCCCAGCCATCTACTAACTC | 316←345 |
580F2 | GTTAGTAGATGGCTGGGAGAAAAAACTTAG | 317→347 |
580R2 | AATATTTAGCCTTCAATTTGTCCCAATCTATG | 437←469 |
580F3 | CTAAATATTTTGATGCAGAAATAGTTAAATGGAGCG | 460→496 |
580R3 | GGCATAATGCTCCCAGTAAACTGGATTTTT | 855←885 |
580F4 | AATCCAGTTTACTGGGAGCATTATGCC | 855→885 |
580R4 | TAAATGTTAGATTGAATCCAGTATGGC | 956←983 |
580F5 | GCCATACTGGATTCAATCTAACATTTA | 956→983 |
580R5 | AGTGTTTATCTAGCAATGTCCATTTTC | 1000←1027 |
580F6 | TGGAAAATGGACATTGCTAGATAAACA | 998→1025 |
580R6 | CGCGGATCCTTATTTATTTTTGTATGAATC | 1071←1092 |
MBOV_RS02825ΔTNASE_3 | ||
580F1 | CGGGGTACCGAAAATGGCACAATTAAG | 79→97 |
580UR | TGGCAATGCAAATTTAGCCTTCAATTT | 530←543, 1027←1040 |
580DF | AAATTGAAGGCTAAATTTGCATTGCCA | 530→543, 1027→1040 |
580R6 | CGCGGATCCTTATTTATTTTTGTATGAATC | 1071←1092 |
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Zhang, H.; Zhao, G.; Guo, Y.; Menghwar, H.; Chen, Y.; Chen, H.; Guo, A. Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity. Int. J. Mol. Sci. 2016, 17, 628. https://doi.org/10.3390/ijms17050628
Zhang H, Zhao G, Guo Y, Menghwar H, Chen Y, Chen H, Guo A. Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity. International Journal of Molecular Sciences. 2016; 17(5):628. https://doi.org/10.3390/ijms17050628
Chicago/Turabian StyleZhang, Hui, Gang Zhao, Yusi Guo, Harish Menghwar, Yingyu Chen, Huanchun Chen, and Aizhen Guo. 2016. "Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity" International Journal of Molecular Sciences 17, no. 5: 628. https://doi.org/10.3390/ijms17050628