Uitdrukking van die rekombinante hepatitis B-virus mantelproteïene in eukariotiese uitdrukkingsisteme

Abstract

The expression of the proteins constituting the envelope of the hepatitis B virus was investigated. A novel human ovarian cancer cell line, UWOV2 (Sf), was tested for its ability to be transfected with and express the hepatits B virus surface antigen genes contained in a mammalian expression vector, using the standard protocols of calcium phosphate/DNA co-precipitation and lipofection. Confluent cell cultures were transfected with the vector which also carried the bacterial neomycin resistance gene and selection was applied with the antibiotic gentamycin. Both transfection procedures resulted in isolated foci of cells that survived the selection after all the cells in control cultures died. None of the cells in these foci survived long enough to be multiplied however, and no further analyses could be carried out it. Positive results were obtained in this laboratory expressing the envelope proteins of the hepatitis B virus in a baculovirus Sf 21-expression system (Strydom 1993 ). A series of fusion protein expression vectors were constructed for the expression of the three hepatitis B virus envelope proteins as His6-fusion proteins that could be isolated using affinity chromatography. The three open reading frames coding for the hepatitis B virus envelope proteins had already been cloned into pSP-vectors. These vectors were used to construct three pBlueBac His baculovirus transfer vectors further used in cotransfections. Recombinant baculovirus clones were prepared by cotransfecting linearized wild type Autographa californica virus genomic DNA with the three different transfer vectors respectively in Sf 21 cell cultures. Recombinant baculovirus clones were prepared and enriched for recombinants using the plaque-assay technique. Sf 21 cell cultures were infected with selected clones and used to quantify the production of HBsAg carrying proteins. An ELISA test kit was used to quantify both the HBsAg in the cell culture supematants as well as in cell lysates and in this way clones that showed the highest HBsAg expression were selected. In all cases the amounts of HBsAg were higher in the cell lysates than in the cell culture supernatants. Cell culture lysates were subjected to SDS-polyacrylamide gel electrophoresis and affinity chromatography on a Ni-NTA column. No fusion proteins could be isolated using this column but proteins with sizes corresponding to that of the envelope proteins of the hepatitis B virus were detected in the total cell culture lysates. In this study it was shown that all three the hepatitis B virus envelope proteins namely the large, medium and major polypeptides could be produced using insect cells infected with recombinant AcNPV clones. It was further shown that these proteins were produced in both the glycosilated and unglycosilated forms. Because the three open reading frames of the envelope protein genes were used seperately, the transcription of these could be compared. The results obtained showed that transcription did not exclusively start at the first initiation codon but in the case of the preS2-S and preS 1- preS2-S open reading frames, also at the second and third in fase initiation codons.