Expression Of Human Herpesvirus 6 U94 ORF In Escherichia Coli, Preparation Of Its Polyclonal Antibodies And Application Of The Protein In Serological Detection

Human herpesvirus 6 (HHV-6) is a beta herpesvirus with a preferentialtropism for CD4~+ T lymphocytes, and was originally isolated from patientswith lymphoproliferative disease and immunosuppression. The primaryinfection with HHV-6 is associated with exanthema subitum(ES), andfollowing primary infection, HHV-6 establishes a latent infection persisting inmonocytes/macrophages and in circulating mononuclear cells in the healthypopulation. Viral reactivation is induced by immunosuppression and can resultin the development of severe diseases. HHV-6 has been associated withseveral pathological conditions, such as complications following solid organand bone marrow transplantation, meningo-encephalitis, infectiousmononucleosis, persistent lymphadenopathy, fulminant hepatitis, autoimmunedisorders, chronic fatigue syndrome, and multiple sclerosis (MS).HHV-6 is classified into two variants: HHV-6A and HH-6B and Bothvariants contain a linear double-stranded DNA genome of approximately 161kbp with 112 potential open reading frames (ORFs). U94 ORF of HHV-6,which has no known counterparts in any of the other human herpesviruses, isexpressed under IE conditions, and encodes a product (RepH6) that ishomologous to the adeno-associated virus type 2 rep geneproduct(RepAAV-2). RepH6, playing a important role in the regulation ofHHV-6 latency, suppresses transformation by H-ras and inhibits transcriptionfrom HIV type 1 long terminal repeat. U94 ORF is transcribed in thecondition of latent infection, whereas other viral mRNA species are notdetected. Furthermore, T cell lines that stably express HHV-6 U94 arepermissive for HHV-6 infection, but viral replication is restricted.In this study, the U94 ORF was amplified by PCR and cloned intoexpression vector pGEX-6p-1. The recombinant protein was expressed in Escherichia coli and purified to immunize New Zealand rabbit for preparationpolyclonal antibody. Also, it was used in serological detection of patients andhealthy adults. The main results are as following:1. HHV-6 cultivation and identificationThe GS standed strain of HHV-6 was grown in CBMCs stimulated withPHA. After 10-12 days, the infected cells exhibited CPE. The DNA of theinfected cells was amplified by nested PCR, and the amplified 287bp outersegment and the 163 inner segment were sequenced. The inner segment couldnot be digested with HindIII. The nucleotide homology analysis indicated thatthe sequence showed 100％identity to HH-6A sequence in Genbank.2. Expression and purification of the recombinant U94 ORF of HHV-6 inEscherichia coliThe U94 ORF was amplified by PCR and cloned into expression vectorpGEX-6p-1. The recombinant protein was expressed in Escherichia coli.Meanwhile experiments on best induced concentration and best induced timeof IPTG showed that the best concentration was lmmol/L and the best culturedtime was 5 hours. Then chromatography was used to purify the expressedRepH6 protein, UV-spectrophotometer showed purfied protein is 0.1mg/ml.The constructed vector which had been identified by PCR, enzyme digestionand nucleotide sequences analysis was transformed into Rosatta. Afterinducing with IPTG, the recombinant protein was purified with GST affinitychromatography. SDS-PAGE and Western blot analysis showed that therecombinant protein was about 40 kD. The purified recombinant proteinRepH6 was used as an antigen to detect specific antibodies in the sera.Different populations were analyzed by enzyme-linked immunosorbent assay(ELISA), including healthy controls, Glioma patients, and immunosuppresspatients. The results show statistically significant differences (P＜0.01)between immunosuppress patients and control groups, in antibody prevalence(87.5 and 53.3％, respectively), and show no statistically significantdifferences (P＞0.05) between Glioma patients and control groups in antibody prevalence (56.3 and 53.3％, respectively). The detection of antibodiesspecific for HHV-6 U94/REP shows that the immune system is exposed to thisantigen during natural infection. The higher prevalence of antibodies toU94/REP suggests that immunosuppress patients and control groups mightexperience different exposures to HHV-6.3. Preparation and identification of polyclonal antibody against therecombinant protein RepH6In the preparation of anti-RepH6 polyclonal antibody, the recombinedRepH6 was inoculated to New Zealand rabbit. After fundamental immunityand strengthening immunity, the serum of rabbit was isolated. Then,thepolyclonal antibodies were prepared by SAS. ELISA showed that its seriumantibody still had combination activity at the titer of 1:10000.