The serum concentration of liver-specific F antigen is a sensitive marker for heaptocellular damage.To develop a serological assay,which could reflect the injuring degree of liver directly,F antigen cDNA of rat liver ...The serum concentration of liver-specific F antigen is a sensitive marker for heaptocellular damage.To develop a serological assay,which could reflect the injuring degree of liver directly,F antigen cDNA of rat liver was obtained by RT-PCR with GenBank based primers,and cloned into plasmid pUCm-T.The amplified fragment was sequenced and compared with F antigen gene in GenBank,result showed that nucleotide sequence of the fragment was the same as that of published F antigen of rat liver.Then the amplified fragment was subcloned into prokaryotic expression vector pET-15b.The recombinant plasmid pET-15b-F was transformed into E.coli BL21(DE3plysS), following induction with IPTG F antigen was successfully expressed.The expressed protein was purified to 90% purity with metal chelate affinity chromatography on Ni-IDA agarose under denature condition.SDS-PAGE and Western blot analysis revealed that the expressed F antigen had a single band of 43 kD and capable of specific binding with anti-F antigen antibody.展开更多
文摘The serum concentration of liver-specific F antigen is a sensitive marker for heaptocellular damage.To develop a serological assay,which could reflect the injuring degree of liver directly,F antigen cDNA of rat liver was obtained by RT-PCR with GenBank based primers,and cloned into plasmid pUCm-T.The amplified fragment was sequenced and compared with F antigen gene in GenBank,result showed that nucleotide sequence of the fragment was the same as that of published F antigen of rat liver.Then the amplified fragment was subcloned into prokaryotic expression vector pET-15b.The recombinant plasmid pET-15b-F was transformed into E.coli BL21(DE3plysS), following induction with IPTG F antigen was successfully expressed.The expressed protein was purified to 90% purity with metal chelate affinity chromatography on Ni-IDA agarose under denature condition.SDS-PAGE and Western blot analysis revealed that the expressed F antigen had a single band of 43 kD and capable of specific binding with anti-F antigen antibody.
