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 inserted or not.
Also to check Hepatitis B Virus DNA orientation in recombinant plasmid, recombinant DNA was digested with XbaI, BamHI, and HincII restriction enzymes. XbaI digestion of recombinant plasmid showed the predicted DNA fragments such as 4297, 1744, and 1641 base pair which we could predict from the characterized gene maps of pUB 110 plasmid and Hepatitis B Virus DNA. In BamHI and HinCII digestion, the predicted DNA fragments were also proved.
From the above experiments, it is evident that Hepatits B Virus genome was cloned and stably propagated in $\underline{Bacillus} \underline{subtilis}$ B.D 170 cells. However, the result of reverse passive hemmagglutination test for the expression of hepatitis B surface antigen in $\underline{Bacillus} \underline{subtilis}$ was negative.
간염 바이러스 전체 유전자(3200 개의 염기쌍) 가 들어있는 pHBV 213 플라스미드를 대장균 ($\underline{E}.\underline{coli}$ C600) 에서 분리하여 EcoRl 제한효소로 자른 후 전기영동장치를 이용하여 간염바이러스 전체 유전자만을 분리해냈다.
고초균 ($\underline{B}. \underline{subtilis}$ B.D.630) 에서 pUB110 플라스미드를 분리하여 EcoRI 제한효소로 자른후에, pUB 110 플라스미드 자체사이의 재봉합을 막기위해 bacterial alkaline phosphatase 로 처리하였다.
위와 같이 처리한 pUB 110 플라스미드와 간염바이러스 전체 유전자를 T4 DNA ligase 로 연결 시킨 뒤, 라이소자임으로 처리하여 나세포 (protoplast) 가 된 고초균 ($\underline{B}. \underline{subtilis}$ B.D.170) 세포속으로 주입하고 재생배지위에서 이틀간 배양하였다.
Kanamycin plate 에서 자란 집락(colony) 으로부터 재조합 플라스미드를 분리한 후, 이들 중 간염바이러스 전체 유전자가 들어있는 재조합 플라스미드를 찾아내어 다시 EcoRI 제한효소로 자르고 전기 영동을하여 고초균내에 간염바이러스 전체 유전자가 성공적으로 cloning 되었음을 확인하였다.
간염바이러스 표면항원에 대한 항체가 부착되어 있는 감작된 면양 적혈구를 이용하여 고초균내에서 간염바이러스 표면 항원의 형질 발현을 조사하였으나 음성반응 결과를 나타내었다.
