| Popis: |
The cloning of glycoprotein H from the Human Herpesvirus 8 (HHV-8) and the testing of its suitability as a diagnostic relevant antigen. The gene of glycoprotein H (gH) from the Human Herpesvirus 8 was successfully amplified using a nested PCR. The gene was cloned in procaryotic and eucaryotic expression vectors. The proof of an eucaryotic expression of the protein of gH in the vaccinia viral system was not achieved. In the bacterial system, the expression of protein parts (F1-F4) succeeded. These fragments covered the whole gene. The length of the fragments differ from 153 to 213 amino acids. It was possible to detect a reaction with Human sera from Kaposis sarcoma patients in Western blotting with bacterial lysate from recombinant E. coli strains. For further immunological characterisation of the gH, antibodies against the F1 part of the gH in rabbits was produced. The immunological reaction of the rabbits antisera was weak which leads to the assumption that the F1 protein in this form is not highly immunogen. The fragement was cleaned up with denaturing conditions, but even after optimization, it was not possible to establish a sensitive and specific ELISA or Western-Blot against this F1-fragment. The results were too weak and not specific enough. Therefore, it was concluded that the gH of HHV-8 does not represent a dominant immunogen of the virus, if expressed in partial proteins. The proof of the gH specific antibodies is only possible with a low sensitivity. A diagnostic use of the gH as ELISA antigen appears as a result questionable. |
