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Updated: Jul 17, 2026
Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line
Published on: September 28, 2022
[Prokaryotic expression and characterization of N-terminal truncated glycoprotein gp85 of Epstein-Barr virus]
Xiang-dong Tu1, Yu-shui Wu, Long-xiang Qiu
1Depertment of Laboratory Medicine, Fuzhou Dongfang Hospital, Fuzhou 350025, China. tuxdmed@126.com
Aim:
To construct a prokaryotic recombinant vector of Epstein-Barr virus (EBV) membrane protein gp85, to express the protein in E.coli and characterize the antigenicity of this non-glycosylated protein.
Methods:
The BXLF2 gene coding 5'-terminal truncated of EBV gp85 was amplified from the EBV strain B95-8 cell line with specific primers. After identification by the restriction digestion with Hind III and Xho I, the PCR product was inserted into the prokaryotic expression plasmid pGEX-5T and confirmed by sequencing. The constructed prokaryotic expression vector pGEX5T-85N was transformed into the competent E.coli BL21. The expressed recombinant protein gp85N was purified by affinity chromatography, characterized by Western blot, and used immunize BALB/c mice. The titer of antiserum from the immunized mice was detected by ELISA.
Results:
Sequencing analysis revealed that the obtained truncated BXLF2 gene was identical to that published in GenBank and successfully cloned into pGEX-5T. SDS-PAGE showed that the expressed recombinant protein was partially soluble with a relative molecular mass of 45,000. ELISA results indicated that the expressed gp85N was recognized by that the anti-gp85 mAb and contiserum with high titer was obtained from the immunized mice.
Conclusion:
The obtained recombinant gp85N with an excellent antigenicity should provide preliminary data for characterization of the antibody produced by the immunized mice.
