Construction and expression of a spliced variant of Epstein-Barr virus bzlf1 and preparation of its polyclonal antibody.
The BZLF1 gene-encoded protein, Zta (EB1, ZEBRA), is a key transcriptional activator of induction of the lytic cycle of EBV. Zta; it contains a basic region with homology to the DNA binding domains of the AP-1 family. In this study, an alternatively spliced BZLF1 (Delta BZLF1) cDNA lacking exon 2, which encodes the DNA-binding domain of Zta, was isolated from B95-8 marmoset cell line releasing EBV. The cDNA was inserted into a prokaryotic expression vector pET-28a+. The His-tagged recombinant protein was overproduced in E. coli BL21(DE3) and purified by nickel affinity chromatography. The purified fraction was characterized by Western blot and MALDI-TOF-MS analysis and used as an antigen to immunize mice. The antibody against Delta Zta can recognize both denatured and natural Zta protein. The Delta Zta protein and its antibody can be used to further investigate its unknown functions.
Qian
Lu
L
Institute of Basic Medical Sciences, Beijing, PR China.
Li
Dan
D
Yang
Zhihong
Z
Hu
Meiru
M
Yu
Ming
M
Shi
Ming
M
Shen
Beifen
B
Guo
Ning
N
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015534:Trans-Activators / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
D000906:Antibodies; C063676:BZLF1 protein, Herpesvirus 4, Human; D018076:DNA, Complementary; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015534:Trans-Activators