Cloning, expression and rapid purification of recombinant chicken interferon-alpha

Phuong Thi Phuong Thao; Le Van Truong; Wen Yi Chia; Dong Van Quyen

doi:10.15625/2615-9023/17614

Author affiliations

Authors

Phuong Thi Phuong Thao Institute of Ecology and Biological Resources, VAST, Vietnam
Le Van Truong University of Science, Vietnam National University of Ho Chi Minh City, Vietnam
Wen Yi Chia University of Nottingham, Malaysia, 43500, Semenyih, Selangor Darul Ehsan, Malaysia
Dong Van Quyen Institute of Biotechnology, VAST, Vietnam

DOI:

https://doi.org/10.15625/2615-9023/17614

Keywords:

Chicken interferon-alpha, E. coli, recombinant, insoluble, expression, immunity.

Abstract

The poultry industry in Vietnam is vulnerable to many viral diseases. Since antibiotics and vaccination provide inadequate protection, it will be beneficial to have alternative immunostimulants to boost non-specific immunity in poultry. Chicken interferon-alpha (ChIFNα) has been described previously with antiviral activity against many pathogens. Therefore, the purpose of this study is to clone and express recombinant ChIFNα in Escherichia coli. The ChIFNα gene was successfully cloned into the pET32a(+) vector, then expressed in the E. coli Rosetta strain. Different expression conditions were tested for the best yield of the expressed protein. Results showed that recombinant ChIFNα was expressed in Rosetta E. coli as inclusion bodies with a yield of 30 mg/100 L culture after induction with
0.5 mM IPTG in 4 hours at 37 ^oC. The recombinant protein was purified using affinity column chromatography under denaturing conditions with the purity > 94%. Western blot analysis indicated that recombinant ChIFNα reacted specifically with its antibody. Further studies will be carried out to characterize the biological activities of recombinant ChIFNα and its application in the poultry industry.

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References

Baeshen M. N., Al-Hejin A. M., Bora R. S., Ahmed M. M. M., Ramadan H. A. I., Saini K. S., Baeshen N. A., Redwan E. M., 2015. Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives. Journal of Microbiology and Biotechnology, 25(7): 953–962.

Costa S., Almeida A., Castro A. & Domingues L., 2014. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: The novel Fh8 system. Frontiers in Microbiology: 5. https://doi.org/10.3389/ fmicb.2014.00063

Davison F., Nair V., 2005. Use of Marek’s disease vaccines: Could they be driving the virus to increasing virulence? Expert Review of Vaccines, 4(1): 77–88.

Jarosinski K. W., Jia W., Sekellick M. J., Marcus P. I., Schat K. A., 2001. Cellular responses in chickens treated with IFN-alpha orally or inoculated with recombinant Marek’s disease virus expressing IFN-alpha. Journal of Interferon & Cytokine Research: The Official Journal of the International Society for Interferon and Cytokine Research, 21(5): 287–296.

Katze M. G., He Y., Gale M., 2002. Viruses and interferon: A fight for supremacy. Nature Reviews Immunology, 2(9), Article: 9.

Kaur J., Kumar A., Kaur J., 2018. Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements. International Journal of Biological Macromolecules, 106:803–822. https://doi.org/10.1016/j.ijbiomac. 2017.08.080

Kong B., Guo G. L., 2014. Soluble expression of disulfide bond containing proteins FGF15 and FGF19 in the cytoplasm of Escherichia coli. PloS One, 9(1): e85890.

LaVallie E. R., DiBlasio E. A., Kovacic S., Grant K. L., Schendel P. F., McCoy J. M., 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology (Nature Publishing Company), 11(2): 187–193.

Ma Y., Long M., Diao A., 2014. Expression, Purification, and Activity Assay of Chicken Interferon-Alpha. In T.-C. Zhang, P. Ouyang, S. Kaplan, & B. Skarnes (Eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012): 741–747. Springer.

Meng S., Yang L., Xu C., Qin Z., Xu H., Wang Y., Sun L., Liu W., 2011. Recombinant chicken interferon-α inhibits H9N2 avian influenza virus replication in vivo by oral administration. Journal of Interferon & Cytokine Research: The Official Journal of the International Society for Interferon and Cytokine Research, 31(7): 533–538.

Mo C. W., Cao Y. C., Lim B. L., 2001. The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and Newcastle disease virus infection. Avian Diseases, 45(2): 389–399.

Read A. F., Baigent S. J., Powers C., Kgosana L. B., Blackwell L., Smith L. P., Kennedy D. A., Walkden-Brown S. W., Nair V. K., 2015. Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens. PLoS Biology, 13(7): e1002198.

Rosano G. L., Morales E. S., Ceccarelli E. A., 2019. New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Science: A Publication of the Protein Society, 28(8): 1412–1422.

Sørensen H. P., Mortensen K. K., 2005. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial Cell Factories, 4(1): 1.

Sun Y. L., Kuan T. C., Lin Y. J., Chou Y. C., Lin C. S., 2010. Construction and expression of rabbit neutrophil peptide-1 gene in Escherichia coli. Annals of Microbiology, 60(2): 329–334.

Tam V., Giang N., Quan N., Binh N., 2014. A study on cloning, expressing and investigating biological activity of recombinant chicken interferon produced from yeast Pichia pastoris. Academia Journal of Biology, 36(1): 216–225.

Thu H. T. V., 2012. Efficiency of recombinant Bacillus subtilis expressed chicken

interferon alpha (ChIFNa- B. subtilis) in prevention of Gumboro disease in chickens. Scientific journal-Can Tho University, 22c: 30–39.

Tomala M., Lavrentieva A., Moretti P., Rinas U., Kasper C., Stahl F., Schambach A., Warlich E., Martin U., Cantz T., Scheper T., 2010. Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. Protein Expression and Purification, 73(1): 51–57.

Tripathi N. K., Shrivastava A., 2019. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Frontiers in Bioengineering and Biotechnology: 7.

Zhao J., Yu H.-Y., Zhao Y., Li F.-H., Zhou W., Xia B.-B., He Z.-Y., Chen J., Jiang G.-T., Wang M.-L., 2019. Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus. Preparative Biochemistry & Biotechnology, 49(2): 192–201.