Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi＇s Sarcoma Associated Herpesvirus orf65

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.     

The Biology of Kaposi＇s Sarcoma-Associated Herpesvirus and the Infection of Human Immunodeficiency Virus

Kaposi sarcoma-associated herpesvirus (KSHV),also known as human herpesvirus 8 (HHV-8),is discovered in 1994 from Kaposi's sarcoma (KS) lesion of an acquired immunodeficiency syndrome (AIDS)patient.In addition to its association with KS,KSHV has also been implicated as the causative agent of two other AIDS-associated malignancies:primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD).KSHV is a complex DNA virus that not only has the ability to promote cellular growth and survival for tumor development,but also can provoke deregulated angiogenesis,inflammation,and modulate the patient's immune system in favor of tumor growth.As KSHV is a necessary but not sufficient etiological factor for KS,human immunodeficiency virus (HIV) is a very important cofactor.Here we review the basic information about the biology of KSHV,development of pathogenesis and interaction between KSHV and HIV.     

Structure variations of TBA G-quadruplex induced by 2＋-O-methyl nucleotide in K＋ and Ca2＋ environments

Thrombin binding aptamer （TBA）, a 15-mer oligonucleo- tide of d（GGTTGGTGTGGTTGG） sequence, folds into a chair-type antiparallel G-quadruplex in the K＋ environ- ment, and each of two G-tetrads is characterized by a syn-anti-syn-anti glycosidic conformation arrangement. To explore its folding topology and structural stability, 2＇-0- methyl nucleotide （OMe） with the C31-endo sugar pucker conformation and anti glycosidic angle was used to selectively substitute for the guanine residues of G-tetrads of TBA, and these substituted TBAs were characterized using a circular dichroism spectrum, thermally differential spectrum, ultra- violet stability analysis, electrophoresis mobility shift assay, and thermodynamic analysis in K＋ and Ca2＋ environments. Results showed that single substitutions for syn-dG residues destabilized the G-quadruplex structure, while single substi- tutions for anti-dG residues could preserve the G-quadruplex in the K＋ environment. When one or two G-tetrads were modified with OMe, TBA became unstructured. In contrast, in Ca2＋ environment, the native TBA appeared to be un- structured. When two G-tetrads were substituted with OMe, TBA seemed to become a more stable parallel G-4 structure. Further thermodynamic data suggested that OMe-substitu- tions were an enthalpy-driven event. The results in this study enrich our understanding about the effects of nucleo- tide derivatives on the G-quadruplex structure stability in different ionic environments, which will help to design G-quadruplex for biological and medical applications.