大鼠肝脏F抗原基因的克隆、表达及产物纯化

肝脏F抗原是一种新型的能直接反映肝损伤程度的血清学标志,它是1968年由美国学者Fravi从小鼠肝中分离出来的[1].F抗原是存在于哺乳动物肝细胞质中的一种水溶性不稳定的单链蛋白质,相对分子量为43kD[2],正常肝组织中的F抗原含量是血清中的1370倍,只有肝细胞被破坏时,F抗原才释放

Cloning, Expression and Purification of Gene Encoding Rat Liver-specific F Antigen

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.