[Prokaryotic expression and characterization of N-terminal truncated glycoprotein gp85 of Epstein-Barr virus].
OBJECTIVE
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.


CONCLUSIONS
The obtained recombinant gp85N with an excellent antigenicity should provide preliminary data for characterization of the antibody produced by the immunized mice.
Tu
Xiang-dong
XD
Depertment of Laboratory Medicine, Fuzhou Dongfang Hospital, Fuzhou 350025, China. tuxdmed@126.com
Wu
Yu-shui
YS
Qiu
Long-xiang
LX
Lian
Yun-zong
YZ
Cheng
Feng
F
Lan
Feng-hua
FH
Zhu
Zhong-yong
ZY
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000906:Antibodies; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins