A DNA fragment of Kaposi's sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus 8 (HHV8), was found by representational difference analysis (RDA) in DNA preparation of Kaposi's sarcoma (KS) tissue from an HIV-infected individual (5). 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 and multicentric Castleman's disease(4, 17). The nucleotide sequence of the virus genome comprises~210 kb and consists of≥90 open reading frames (ORFs) (14). By now, there have been many studies around the world investigating the distribution and infection of KSHV in different areas. KSHV is found to have a worldwide occurrence but infection rates vary according to a combination of geographic and behavioral risk factors (15). Seroprevalence of KSHV is shown to be 25% higher in African countries, whereas in the United States, Asia, and Western Europe it is lower than 10% (3, 9, 19). Several methods have been implemented for detecting KSHV, mainly based on serological and molecular diagnosis (6, 7, 12, 16). At least 4 different serologic assays have been utilized to detect the HHV8 antibodies; immunofluorescent assays (IFA), enzyme-linked immunosorbent assays (ELISA), Western blot and immunohistochemistry (IHC). For screening a large amount of sera, the ELISA method is always used in the epidemiologic research. The quality of antigen is essential in ELISA assays; therefore, purified antigen with high quality should be used.
orfK8.1 was identified to be located at nucleotide positions 76 214-76 994 within the HHV8 genome, orfK8.1 codes for a 35 to 37 kDa protein that is most likely located within the envelope of the virus and appears to be a major target for human antibodies (13). In this study, the orfK8.1 was cloned and expressed as a recombinant protein in E. coli., and our data indicates that it possesses good antigenicity and specificity in ELISA detecting KSHV using sera.
A 538 bp fragment was obtained by PCR and cloned into pGMT-easy vector and sequenced. The orfK8.1 gene fragment was then subcloned into the pQE-80L vector; the recombinant plasmid was named as pQE80L-orfK8.1. After being induced by IPTG for 6 h and identified by SDS-PAGE electrophoresis, proteins at about 26 kDa were expressed in BL21 (DE3) strain transformed by pQE80L-orfK8.1 (Fig. 1). Further separation of supernatant and precipitation indicated that the concentration was very high in the precipitation of the lysed BL21 (DE3) transformed by pQE80L-orfK8.1. High purity recombinant precipi-tation protein was attained after Ni2+-nitrolotriacetic acid resin affinity chromatography purification; analysis using the bandscan software indicated that the purity of the recombinant ORFK8.1 protein was about 95% (Fig. 1).
Figure 1. Expression and purification of KSHV ORFK8.1 protein. 1, The cell lysate of pQE80L-orfK8.1/BL21(DE3) before IPTG induction; 2, The cell lysate of pQE80L-orfK8.1/BL21(DE3) after IPTG induction; 3, The supernatant of the lysed pQE80L-orfK8.1/BL21(DE3) after IPTG induction; 4, The precipitate of the lysed pQE80L-orfK8.1 /BL21 (DE3) after IPTG induction; 5 and 6, Purified ORFK8.1; M, Protein marker.
The total protein of lysed BL21 (DE3) transformed by pQE80L-orfK8.1 and the purified recombinant ORFK8.1 were electrophoresed by SDS-PAGE and transferred to a PVDF membrane. The membrane was coated with the KSHV positive serum or negative serum. A specific 26 kDa band occurred in the lanes of the total and purified protein of lysed BL21 (DE3) transformed by pQE80L-orfK8.1 when proteins were incubated with serum from KS patient; no band was seen when proteins were incubated with serum from healthy blood donors (Fig. 2). The result indicates that the recombinant ORFK8.1 has very strong specificity and antigenicity.
Figure 2. Western blot of recombinant ORFK8.1 protein. A: Reacted with serum from KS patient. B: Reacted with serum from blood donor. 1, The cell lysate of pQE80L-orfK8.1/BL21 (DE3) before IPTG induction; 2, The cell lysate of pQE80L-orfK8.1/ BL21(DE3) after IPTG induction; 3, Purified ORFK8.1.
The antigenicity of the recombinant ORFK8.1 was also assessed by ELISA. The recombinant ORFK8.1 was used as antigen to coat the ELISA plates; KSHV positive and negative serum were used as the primary antibody. The ELISA result showed that the optimal antigen concentration was 5 μg/mL (Fig. 3A) and the optimal dilution of the serum was 1: 200 (Fig. 3B). The result confirmed the antigenicity and specificity of the recombinant ORFK8.1 as a diagnostic antigen for KSHV.
To examine the KSHV serostatus in the studied subjects, KSHV lytic antigen ORFK8.1 was used to detect specific antibodies. To evaluate the overall specificity and the detection ratio of the assays, 20 classical KS patients provided and confirmed by the Laboratory of Xinjiang Endemic and Ethnic Diseases (Shihezi University) were examined who would usually be 100% positive for ORFK8.1 antibody (10) and 50 healthy subjects which were characterized previously (8) were also included as control. Overall, the KSHV serological assay using recombinant ORFK8.1 could determine all the 20 KS sera as KSHV positive and thus had a combined specificity of 100%.
Using the ELISA assay, KSHV seroprevalence in the general population in Hubei Province was examined. Of 560 subjects, 38 (6.80%) were KSHV seropositive. Among them, 17(5.36%) were male, 21(8.64%) were female. Analysis of the subjects by age showed that KSHV seroprevalence changed with subject age. Under 20, 20-50, and over 50 have 6.06%, 7.59% and 5.80% KSHV seroprevalence respectively (Table 1).
Table 1. KSHV seroprevalence in Hubei general population