Int J Med Sci 2017; 14(11):1072-1079. doi:10.7150/ijms.20417
Construction and Identification of Recombinant HEK293T Cell Lines Expressing Non-structural Protein 1 of Zika Virus
1. School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, Guangdong, China;
2. Institute of Comparative Medicine and Center of Laboratory Animals, Southern Medical University, Guangzhou, Guangdong, China;
3. Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, USA;
4. Beijing Minhai Biotechnology CO. LTD, Beijing, China.
* These authors contributed equally to this work.
Background: Zika virus (ZIKV) infection has become a major public health problem all around the world. Early diagnosis of Zika infection is important for better management of the disease. Non-structural protein 1 (NS1) is a potential biomarker for ZIKV infections. The purpose of this study was to produce the ZIKV NS1 protein for establishing serological diagnostic methods for ZIKV.
Methods: The cDNA fragment encoding a chimeric protein composed of murine Igκ signal peptide, NS1 and histidine tag was synthesized and cloned into the lentiviral expression vector pLV-eGFP. The resulting expression vector pLV-eGFP-ZIKV-NS1 was packaged and transduced into human embryonic kidney (HEK) 293T cells and clonal cell lines with NS1 gene were generated from the tranduced cells by limiting dilution. Over expressed recombination NS1 (rNS1) fusion protein was purified by nickel affinity chromatography. Mice immunization and enzyme-linked immunosorbent assay (ELISA) were carried out to evaluate the immunogenicity of rNS1.
Results: Western blot analysis revealed that the reconstituted cells stably expressed and secreted high levels of approximately 45-kDa NS1, and no significant changes were observed in green fluorescent protein (GFP) fluorescence ratio and fluorescence intensity. The scanned gels showed that the purity of the purified rNS1 was 99.42%. BALB/c mice were then immunized with purified rNS1 and a high level of antibodies against NS1 was elicited in the mice.
Conclusion: Overall, recombinant NS1 proteins were successfully purified and their antigenicity was assessed. Immunization of mice with recombinant proteins demonstrated the immunogenicity of the NS1 protein. Thus, the generated recombinant NS1 can be potentially used in the development of serological diagnostic methods for ZIKV.
Keywords: ZIKV, Non-structural protein 1, HEK293T cell, Secretory expression, Purification.
Liu J, Wan P, Li Q, Li X, Li A, Chen H, Li J, Liang W, Zheng H, Gu W, Li H. Construction and Identification of Recombinant HEK293T Cell Lines Expressing Non-structural Protein 1 of Zika Virus. Int J Med Sci 2017; 14(11):1072-1079. doi:10.7150/ijms.20417. Available from http://www.medsci.org/v14p1072.htm