Cloning and Expression of Human IL - 10 cDNA and Biological Activity of Expressed Product

Objective To express recombinant human IL - 10 in E. coli. Methods A hIL - 10 DNA fragment, with a length of about 500bp, was amplified from the RNA of leukemia cell strain K562 by RT - PCR and cloned to plasmid pSK( + ) ,and the cloned DNA fragment was sequenced. The recombinant plas-mid pSK/IL- 10 was digested with EcoRI and BamHI, then hIL- 10 fragment was isolated and inserted to the corresponding restriction site on procaryotic expression vector pBV220.The recombinant plasmid PBV/IL - 10 was identified by enzymogram and transformed to E. coli, then expressed by induction at 42℃ .The expressed product was identified by SDS-PAGE and Western blot and renaturalized with glutathione buffer, and the inhibitory effect of it on the production of IFN - γ in PBMC was detected by RT - PCR. Results The length of DNA fragment amplified by RT - PCR was consistent with that of hIL - 10 cDNA. DNA sequencing of pSK/ IL- 10 revealed that the cloned DNA sequence was identical to that of reported hIL- 10 cDNA.SDS - PAGE proved that the expressed product, with a relative molecular weight of 18000,contained about 20% of total somatic protein. Western blot showed that the recombinant protein could specifically bind to anti-hIL-10 antibody and,after being renaturalized, could inhibit the production of IFN-γ in PBMC significantly. Conclusion A recombinant bacterial strain for expressing hIL - 10 with biological activity was successfully constructed.