Kaposi's sarcoma-associated herpesvirus, also called human herpesvirus 8 was first found in 1994 by Chang et al from one AIDS-KS (Kaposi's sarcoma) patient by representative differentiation assay (3). Since then, the causal link between KSHV and KS has been well established. KSHV is consistently found in all forms of KS: (classic, endemic, AIDS associated and transplantation associated). KSHV has also been observed to be associated with primary effusion B cell lymphoma (2) and multicentric Castleman's disease (10). KSHV is a large double stranded DNA virus with a size of about 210 kb; it encodes approximately 90 open reading frames (orf). To date scientists around the world have performed a number of investigations concerning the distribution and infection of KSHV in different regions. KSHV is found to have a worldwide occurrence but infection rates vary according to a combination of geographic and behavioral risk factors (10). Seroprevalence of KSHV is shown to be higher than 25% in African countries, whereas in the United States, Asia, and Western Europe is lower than 10% (1, 6, 13). Many different methods have been set up for detecting KSHV, mainly including serologic and molecular diagnosis (4, 5, 8, 11). 4 different serologic assays have been used to detect the HHV-8 antibodies; immunofluorescent assays (IFA), enzyme-linked immunosorbent assays (ELISA), Western blot and immunohistochemistry (IHC). The antigen used for ELISA can be recombinant antigen, the whole virus or the synthetic peptide but quality is important and purified antigen of high quality should be used. However, the ELISA method is simple and so it is the optimal choice for screening a large amount of sera for epidemiological research.
KSHV orf65 encodes for a small capsid protein and the recombinant orf65 has very good antigenicity and specificity, and so it represents a good choice for in the KSHV epidemiological research (7). In this study, we have cloned the c-terminal of orf65, and expressed it as recombinant protein in E.coli, and our data indicates that it possesses very good antigenicity and specificity.
Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65
- Received Date: 06 December 2007
- Accepted Date: 06 January 2008
Abstract: To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA