Cloning,sequence analysis and biological characterization of human recombinant IL 12

Objective:Cloning IL 12p40 from Chinese and constructing human recombinant IL 12 expression vector.Methods:By using RT PCR to clone IL 12 p40cDNA from the mRNA extracted from cord blood dendritic cells(DC) of Beijing newborns' and then sequenced.Expression vectors encoding the IL 12 subunits p35,p40 and IL 12 bicistronic expression vector were constructed by using pCDNA3.1 and pLXPXSN,and then transfected human heptocellular carcinoma(HCC) cell line by lipofection.Results:IL 12 cloned from DC of Beijing has unique structure in codon 210,being GCC(Ala).The 239 and 291 codons are the same as CLMF p40 but different from NKSF p40.Transfection with expression vectors encoding either the p40 or the p35 of hIL 12 to HCC cell line showed that only in the case of co transfection with both vectors in equivalent amount,the supernatant of the transfected cells showed biological activity of IL 12.Subsequently,the bicistronic retroviral vector encoding both p40 and p35 of hIL 12 was transfected the replicating HCC cell line.Following selection,the supernatant of the transduced cell colonies showed prominent biological activity in stimulating the proliferation of lymphoblasts.Western blot showed the appearance of the characteristic 70 kD band.Conclusion:The above data support the hypothesis that the p40 gene of human IL 12 may be polymorphic.