HHV8Detection In Blood Donors And HHV-8Recombined Antigen Expression

Background and purposeHuman herpesvirus8(HHV-8) is a new type human herpesvirus isolated and identified in1994. It was initially called Kaposi’s sarcoma-associated herpesvirus(KSHV). HHV-8belongs to human herpesvirus subfamily and DNA virus. It is considered to be pathogenic factor of kaposi sarcoma. Beside HIV/AIDS, HHV-8also associated with multicenter Castleman’s disease, primary effusion lymphoma and a variety of diseases. HHV-8spreads mainly through saliva, sex and blood. It usually exists in latent infections state in the body and the virus come into replicate stage and cause diseases when stimulated by some factors. So far, two main methods are used for HHV-8identification. One is DNA detection using PCR, with the problem of high false positive; the other is ELISA assay. But the sensitivity reduced, mission ratio enhanced and reagent antigen depends on import, for the single antigen. The epidemiological survey shows that HHV-8has obvious regional differences in the infectious situation of population except patients of related disease, but infectious situation of HHV-8in the general population of our country is rarely reported. To understand the infectious situation of HHV-8in general population of central China and improve the sensitivity of HHV-8ELISA diagnostic reagent and provide domestic antigen, RT-PCR and ELISA assays were used and HHV-8infection situation of480cases of unpaid blood donors in Luoyang area was tested and analyzed. Over-lap PCR was used to obtain ORF50and ORF73fusion gene fragment and construct the prokaryotic expression vector. Expression of recombinant fusion protein was induced and HHV-8recombinant fusion protein was identified. Method1. Collected480specimens of Luoyang blood donors in2011, each specimen contains0.5mL anticoagulant blood and0.5mL serum.2. Using HHV-8double antigen sandwich enzyme-linked immunoassay reagent to identify antibody situation of480cases of unpaid blood donors’ serum.3. Isolate the lymphocytes and extract DNA of480anticoagulant specimens; Using RT-PCR to identify HHV-8DNA.4. The primers of ORF50and ORF73genes were designed and synthesized. Using HHV-8positive serum DNA as template. ORF50and ORF73genes were obtained by PCR. Connect the two gene fragments and gain the fusion gene of ORF50and ORF73.5. We gained positive clones recombinant pGEM-T-ORF50-ORF73, after recombining fusion gene of ORF50and ORF73into T vector pET-28a; PCR screened positive clones with T7/SP6.6. Using double enzyme HindⅢ and BamHI to digest pGEM-T-ORF50-ORF73and recycling ORF50and ORF73fusion gene fragment and recombine it in the prokaryotic expression vector pET-28a, using PCR, HindⅢ and BamHI double enzyme digestion and DNA sequencing identification to get the subclone recombinant pET-a-ORF5028-ORF73.7. The fusion proteins ORF50-ORF73were expressed by induction with IPTG and then were purified.8. Using Western blot to identify the recombinant fusion protein; serum of blood donors were detected after recombinant fusion protein enzyme labeled plate.Results1. ELISA results:In480serum specimens,31cases were HHV-8antibody positive, positive rate was6.5%(31/480).2. PCR Results:In480serum specimens,32cases were HHV-8DNA positive, positive rate was6.7%(32/480);31cases were HHV-8antibody positive in serum, positive rate was6.5%. All of the31cases of HHV-8antibody positive specimens are HHV-8DNA positive, only one case was HHV-8DNA positive but antibody negative. HHV-8positive rate was detection by PCR and ELISA, There was no statistically significant difference (P>0.01).3. The difference of HHV-8infection rate in different gender, age group unpaid donors has no statistical significance (P>0.05).4. Using PCR amplification to get designed ORF50and ORF73gene fragments, using over-lap PCR amplification to get ORF50and ORF73gene which was connected with fragment gene of597bp.5. ORF50and ORF73fusion gene reconbined in T vector, hundreds of white bacterial colonies were got after conversion.PCR amplification appraisal get positive clone recombinant pGEM-T-ORF50-ORF73.6. Subclone recombinant pET-28a-ORF50-ORF73was consistent with expectations after PCR, double enzyme identification and sequencing identification. It was proved that the prokaryotic expression of ORF50and ORF73fusion gene vectors were successfully constructed.7. SDS-PAGE showed induced ORF50and ORF73recombinant fusion protein was about21Kd and was consistent with expectation.8. Western blot result showed ORF50and ORF73recombinant fusion protein specifically combine with HHV-8positive serum. Recombinant fusion protein; Recombinant fusion protein packaged ELISA plate and then reaction with serum of blood donors and detected HHV-8infection status, the result was consistent with HHV-8ELISA kit.Conclusions1. There is a certain proportion of HHV-8patients in the general population of central China. The dangers of HHV-8transfusion transmission worth further discussion.2. The method about preparation of recombinant ORF50and ORF73fusion antigen of HHV-8was initially established and provides a experiment basis to comparison and evaluation of the fusion antigenic specificity and sensitivity.