RESUMEN
Objective To prepare a high‐titer rabbit specific serum antibody against Kaposi′s sarcoma associated herpesvirus (KSHV) ORF65 capsid protein and identify the specificity of serum antibody .Methods Artificial synthetic peptide of ORF65 pro‐tein was emulsified with Freund adjuvant .4 rabbits were immunized with the prepared antigen by subcutaneous injection at various sites of skin of back and jaw once every two weeks .Immunization was carried out in total 4 times .The serum of the immunized rab‐bits was collected at a week after the last immunization .The titer of rabbit anti‐serum was assayed by ELISA .Specificity of the rab‐bit anti‐serum was analyzed by immunofluorescence assay and Western blot .Results The immunized rabbits produced high‐titer se‐rum antibody after total immunization .The highest titer of anti‐serum against ORF65 protein peptide was 1∶12 800 .The results of Immunofluorescence assay showed that antibody was binded in plasma of BCBL‐1 cell mostly ,which was consistent with the expres‐sion location of ORF65 in BCBL‐1 cell .Subsequently ,the data of Western blot revealed a specific band about 21 kD which accorded with the size of ORF65 protein .Meanwhile ,the expression of ORF65 in TPA treated BCBL‐1 cells was higher than the control cells ,which was consistent with the expression characteristics of lytic protein .Conclusion High‐titer specific rabbit serum antibody against KSHV capsid ORF65 antigen could be successfully prepared by rabbits immunization with ORF65 protein peptide .The pre‐pared antibody could be revealed immune reaction specificity with KSHV ORF65 protein .
RESUMEN
Objective To construct the prokaryotic expression plasmid of HHV-8 fusion antigen for diagnosis of HHV-8 infec-tion .Methods The combined fragment ORF59 ,ORF65 and K8 .1 by fusion PCR was integrated into pQE-80L and transfected into E .coli DH5α.Fusion protein was induced to express by IPTG .SDS-PAGE and Western blot were employed to detect the fusion protein .Fusion protein was used to detect serum of blood donors .Results The combined plasmid pQE-80L-ORF59-ORF65-K8 .1 was constructed successfully after verifying by restriction enzyme digestion and sequencing .The fusion protein was about 24 KD and could be specific combined with HHV-8 positive serum .The fusion protein had the same result to detect HHV-8 with the HHV-8 ELISA kit .Conclusion Fusion protein we construct can be used as diagnosis antigen to detect HHV-8 of blood donors and common people .
RESUMEN
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.