Screening of CHO cell clones expressing histidine-tagged major S hepatitis B surface protein using a semi-quantitative PCR protocol.
Different mammalian cells have been used successfully for the expression of the hepatitis B surface antigen (HBsAg). The patterns of expression of the HBsAg seem to depend on the cell type and the number of gene copies integrated into cellular genome. The expression of an HBsAg fused to histidine tag (His-HBsAg), had not been reported in mammalian cells. This paper describes a semi-quantitative polymerase chain reaction (PCR) employed to investigate the patterns of expression of His-HBsAg in stably transfected Chinese hamster ovary (CHO) cell clones. pcDNA4CR20, a mammalian expressing vector, encoding His-HBsAg, was constructed. The correlation between His-HBsAg expression and the number of integrated copies of the HBsAg gene into cell clones genome was evaluated by the semi-quantitative PCR, with limit dilutions of the genomic DNA as template. The results show a positive correlation between the expression levels of His-HBsAg with the number of the HBsAg gene copies integrated into CHO cell clones. The approach of a semi-quantitative PCR proved to be a good and non-expensive alternative strategy to analyze the patterns of expression of integrated genes into CHO cells genome.
Vianna
Carlos Otávio Alves
CO
Laboratory of Virological Technology, Department of Technological Development, P.O. Box 926, Bio-Manguinhos, Fiocruz, Rio de Janerio, Brazil.
da Silva e Mouta Junior
Sergio
S
de Oliveira da Silva
Gerson
G
da Silva Freire
Marcos
M
de Moraes
Marcia Terezinha Baroni
MT
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D018628:Gene Dosage; D016678:Genome; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection; D014773:Virology / Q000379:methods*; D016662:Virus Integration
D006514:Hepatitis B Surface Antigens; D006639:Histidine