Molecular cloning and expression of woodchuck granulocyte-macrophage colony stimulating factor.
Granulocyte-macrophage colony stimulating factor (GM-CSF) has immunoregulatory and antiviral effects, and may thus be promising for the treatment of chronic hepatitis B. Using woodchuck hepatitis virus (WHV)-infected woodchuck as an animal model to test the efficacy and safety of GM-CSF on the therapy of chronic hepatitis B, woodchuck GM-CSF will be required due to the apparent species-specific activity of GM-CSF. The cDNA of woodchuck GM-CSF was cloned using reverse transcription-polymerase chain reaction (RT-PCR) with primers deriving from highly conserved regions of GM-CSF genes from other species. The deduced amino acids, including the signal peptide, is 138 in length and its identities to human, murine, canine and bovine GM-CSFs are 63, 49, 63, and 63% respectively. The genomic DNA of woodchuck GM-CSF was also cloned by PCR. Its organization is highly homologous to that of human and murine GM-CSF genes, consisting of four exons and three introns. Cloned woodchuck GM-CSF was expressed transiently in 293T cells. The recombinant protein expressed was found to stimulate the growth and differentiation of woodchuck bone marrow cells, indicating the protein expressed by the cloned gene is functional. These results pave the way for future studies on the potential role of GM-CSF for the treatment of chronic hepatitis B by using this animal model.
Wu
H L
HL
Hepatitis Research Center, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan.
Chen
P J
PJ
Lin
H K
HK
Lee
R S
RS
Lin
H L
HL
Liu
C J
CJ
Lee
P J
PJ
Lee
J J
JJ
Chen
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004195:Disease Models, Animal; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000627:therapeutic use; D017919:Hepatitis B Virus, Woodchuck / Q000378:metabolism; D019694:Hepatitis B, Chronic / Q000188:drug therapy; D006801:Humans; D008392:Marmota; D051379:Mice; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D014162:Transfection
D018076:DNA, Complementary; D016178:Granulocyte-Macrophage Colony-Stimulating Factor