Die inhibisie van die hepatitis B-virus oppervlakantigeen deur van antisin tegnologië gebruik te maak

Abstract

The Hepadna virus family is a major annual cause of suffering and loss of life. The Hepatitis B virus is one of the most important causes of this phenomenon, since it is endemic in large parts of the world. This virus was only discovered late in the twentieth century, and its clinical impact was only discovered much later. Infection with the Hepatitis B virus is the cause of liver cirrhosis and recurring infections can lead to the formation of hepato cellular carcinoma. These reasons were the motivation for this study. Firstly the ability of an antisense oligonucleotide to inhibit the production of the major surface antigen (HBsAg) was investigated. This protein is viewed as one of the causes of liver cirrhosis and hepato cellular carcinoma. A 15 mer antisense oligonucleotide, complimentary to the initiation region of the viral S gene DNA, was used. The antisense oligonucleotide was transferred into a hepato cellular carcinornic cell line in different concentrations by means of an appropriate transfection technique. The expression of the S gene was inhibited to over 30% over a time span of five days by treating the cells with 20μM of the antisense oligonucleotide. The inhibition of this S-gene protein could however, not be achieved for time spans longer than five days. It was therefore decided to construct an expression vector to transfect the same cell line, in order to obtain inhibition of a more permanent nature. An appropriate expression vector was manipulated by using biotechnological techniques, in such a way that the Hepatitis B virus S gene was incorporated into the antisense orientation. The expression vector was transferred into the hepato cellular carcinornic cell line by using the same techniques as for the oligonucleotides. The levels of the S gene expression of the cell line was measured 49 days after transfection and levels of inhibition as high as 50,6% were obtained.

Parts of this study were presented at the following national scientific meetings: M. BOTHA, T.S. KLERCK & W.J. DE WET (1995), Therapeutic use of antisense oligo( dN)s for the inhibition of the Hepatitis B virus surface antigen (HBsAg). (Paper delivered at the Thirteenth Congress of the South African Biochemical Society, University of the Orange Free State, Bloemfontein, South Africa). M. BOTHA, T.S. KLERCK & W.J. DE WET (1994), Therapeutic use of antisense technology for the inhibition of the Hepatitis B virus surface antigen (HBsAg). (Paper delivered at the Eighth Biennial Congress of the South African Society for Microbiology Rhodes University, Grahamstown, South Africa). R. PFAHL, M. BOTHA, T.S. KLERCK & W.J. DE WET (1994), Therapeutic oligodeoxynucleotides [Oligo(dN)] for specific inhibition of the Hepatitis B virus surface antigen. (Paper delivered at the Eighth Biennial Congress of the South African Society for Microbiology Rhodes University, Grahamstown, South Africa).