Subcloning of hepatitis B virus genome(ayw) into bacillus subtilis

Abstract

As the Hepatitis B Virus can not grow in cell culture at the present time and normally infects only man and apes, the only available viral source has been dependent on the limited amounts of material from the plasma of infected patients. Gene cloning of Hepatitis B Virus DNA into bacteria and eukaryotic cell lines was tried for large-scale production of Hepatitis B Virus DNA and viral antigens for diagnostic purpose. Although $\underline{Escherchia} \underline{coli}$ has been used as the host for expressing cloned genes, $\underline{Bacillus} \underline{subtilis}$ has been considered as an attractive candidate for the development of a cloning system with various advantages. Hepatitis B Virus DNA was inserted into a bacillus vector, pUB110, to check the possibility of cloning of Hepatitis B Virus DNA in $\underline{Bacillus} \underline{subtilis}$ and to express genes coding for viral proteins in $\underline{Bacillus} \underline{subtilis}$. Plasmid pUB110 (4.5Kb) was digested by EcoRI restriction enzyme and treated with bacterial alkaline phosphatase to inhibit the self-ligation of EcoRI-digested pUB110 plasmid. Hepatitis B Virus DNA (3.2Kb) eluted from EcoRI-digested pHBV213 plasmid which contains a single entire genome of Hepatitis B Virus in pBR 322 plasmid, was ligated with EcoRI-Bacterial alkaline phosphatase treated pUB 110 by T4 DNA ligase at 14℃ for 12 hours. $\underline{Bacillus} \underline{subtilis}$ B.D 170 cells (as a host cell) were protoplsated by treating lysozyme (1mg/ml) and the protoplasted cells were transformed with the above ligate mixture. The transformed cells were grown on regeneration media for 2 days. The sucessful transformation was checked by rapid plasmid isolation from the each transformant (Kanamycin-LB-Agar plate, 5ug/ml). Recombinant plasmid DNA which showed an increase in length on 1% agarose gel electrophoresis, was again digested with EcoRI enzyme and checked on 1% agarose gel whether Hepatitis B Virus DNA (3.2Kb) was ins...