Expression, purification and immunogenicity analysis of the extracellular domain of tick-borne encephalitis virus envelope glycoprotein
Journal Title: Journal of Air Force Medical University - Year 2023, Vol 44, Issue 6
Abstract
Objective To construct prokaryotic expression plasmid of the extracellular domain of envelope glycoprotein E of tick-borne encephalitis virus (TBEV) Senzhang strain, and induce expression, purification and preliminary analysis of its immunogenicity in Escherichia coli. Methods The gene sequence of extracellular domain of E protein of TBEV Senzhang strain was obtained from Genbank, then synthesized by the company after codon optimization, and cloned into pET28a vector by double endonuclease digestion to obtain the recombinant plasmid pET28a-TBEV-Eecto. After restriction endonuclease digestion and sequencing analysis, the correct recombinant plasmid was transformed into BL21 (DE3), which was induced by 1 mmol / L isopropyl-β-D-thiogalactopyranoside. The expression form of recombinant E protein in extracellular domain was analyzed by SDS-PAGE. The purified inclusion bodies were denatured and dissolved with 6 mol / L guanidine hydrochloride, and then renatured with NDSB-201 buffer, which was concentrated via ultrafiltration, purified by nickel ion affinity column chromatography, and identified by Western blotting. TBEV-Eecto protein was immunized to BALB / c mice, and Western blotting and immunofluorescence assay were used to analyze the specificity of immune serum. Results The fragment size of recombinant plasmid pET28a-TBEV-Eecto was consistent with the expected size, and the sequencing analysis was correct. The recombinant protein TBEV-Eecto was mainly expressed in the form of inclusion bodies. The purified protein was obtained after guanidine denaturation, ultrafiltration and nickel ion column affinity chromatography. SDS-PAGE and Western blotting analysis showed that its size was consistent with the expected size. The purified TBEV-Eecto protein could specifically recognize the eukaryotic expression of TBEV-PrME protein in mouse serum, suggesting that TBEV-Eecto has good immunogenicity. Conclusion The prokaryotic expression plasmid of E protein in extracellular domain of TBEV Senzhang strain is successfully constructed and expressed in Escherichia coli. The purified TBEV-Eecto protein has good immunogenicity, which provides a basis for serological diagnosis and subunit vaccine development of tick-borne encephalitis.
Authors and Affiliations
CHEN Yang, ZHANG Jian, CHEN Hua, XUE Pan, WU Fuxing, YUAN Mingcheng, YUAN Ruodong, DONG Yangchao,YE Wei, YE Chuantao, LEI Yingfeng
Keywords
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