Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells.
OBJECTIVE
To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably.


METHODS
Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1-S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind III/Not I followed by ligation with pRc/CMV, or BamHI/EcoR I followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSG5UTPL/Flag plasmid vectors with T4 DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were Compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418.


RESULTS
Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preS1-preS2S and preS1S encoding genes, determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins. Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen.


CONCLUSIONS
Eight recombinant plasmids expressing S, M, L or preS1S proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells, the vector pRc/CMV is superior to pSG5UTPL/Flag, and pRc/CMV-S and pRc/CMV-MS are the most efficient in the pRc/CMV clones. SP2/0 cells stably expressing HBsAg are established, and may be used as target cells for evaluating the CTL activity of a DNA vaccine in vitro.
Qin
Shan
S
Key Laboratory for Molecular Biology of Infectious Diseases of Sichuan Province, West China Hospital, Sichuan University, Chengdu 610041, China. shanqin@hotmail.com
Tang
Hong
H
Zhao
Lian-San
LS
He
Fang
F
Lin
Yong
Y
Liu
Li
L
He
Xiao-Mei
XM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D014162:Transfection
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen