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Herpes simplex virus type 1 (HSV-1), a typical alphaherpesvirus, is a common human pathogen that is associated with infections of the mucocutaneous membranes, brain, and internal organs of infected neonates[8]. The HSV-1 genome is a 152-kilobase (kb) linear duplex DNA molecule, and 84 open reading frames were expressed in the infected cells. Expression of the viral genes occurs in a coordinately activated cascade that consists of the sequential expression of immediate-early (IE), early (E), and late (L) genes[10]. Infected-cell protein 27 (ICP27), a 63 kDa immediate-early regulatory phosphoprotein, is an essential, highly conserved protein and has been extensively studied[16]. ICP27 is involved in various steps of HSV-1 gene regulation as well as in the shut-off of host gene expression during infection. Recently, many novel functions have been reported for the ICP27 protein, including inhibition of the IFN-1 signaling[6], regulation of the viral mRNA translation[3] and determining the composition of HSV-1 virions[17].
In this study, we constructed a recombinant expression plasmid that drives expression of the HSV-1 ICP27 fused with His6. The recombinant protein His6-tagged ICP27 was expressed in E. coli BL21 (DE3) cells. His6-tagged ICP27 was purified by single-step immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin and applied to raise the antiserum in rabbit. The antiserum, shown to be sensitive and specific by Western blot and immunofluorescence analysis, can be applied to further characterize the biological function of ICP27 during HSV-1 infection.
Herpes Simplex Virus Type 1 ICP27 Protein: Its Expression, Purification and Specific Antiserum Production
- Received Date: 11 December 2009
- Accepted Date: 01 February 2010
Abstract: Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27’s biological function during HSV-1 infection.