Eukaryotic Expression Of Recombinant Human Interleukin-29 Variants And Inhibition Of Tumor Cells Proliferation Analysis

h IL-29, also known as IFN-λ1, is a new cytokine discovered in 2003. h IL-29 binding with its receptor complex(consisting of IFN-λR1and IL-10R2 subunit) to activate identical JAK-STAT signal transduction pathways. The biological effects of h IL-29 include the antitumor proliferation, antiviral and immunoregulatory. h IL-29 receptors are expressed in a tissues-specific manner. h IL-29 should be further developed as a new gene engineering drugs with little side-effects.Comparison of tertiary structure of hIL-29 with crystal structure of hIL-28 B, three amino acid residues of hIL-29 were different from h IL-28 B at the positions in helices A: 33 Lys, 35 Arg, 43 Lys and in helices F 143 Ala, which corresponded to 34 Arg, 36 Lys, 44 Leu, 144 Pro in h IL-28 B. Based on the corresponding residues of of h IL-28 B, hIL-29 mutants were created at positions 44 Leu and 143 Ala by site-directed mutagenesis, then analyze the antitumor proliferation activity of h IL-29 variants.A mutant gene of h IL-29 was amplified by megaprimer PCR. A recombinant plasmid was constructed and transformed into Pichia pastoris GS115, the recombinant protein rh IL-29K43 L and rhIL-29A143 P was induced and purified, HPLC analyses showed that their purity were 98.17% and 97.83%, respectively. The purified recombinant protein has relative molecular masses of about 23.0 kDa on SDS-PAGE profilet, which was showed specific reaction with goat anti-human IL-29 polyclonal antibody in Western-Blotting.The effect of recombinant protein rhIL-29K43 L and rhIL-29A143 P on proliferation of tumor cell lines(human gastric carcinoma SGC7901, human colonic carcinoma HCT8, human hepatic carcinoma BEL7402, human lung adenocarcinoma A549 and human acute monocytic leukemia THP1) was detected by Cell Counting Kit-8 reagent. The results showed that the rh IL-29K43 L and rhIL-29A143 P had anti-proliferation effect and its effect was stronger at high dose group than that of wild type rhIL-29,the proliferation inhibition ratios of rh IL-29K43 L on the above tumor cells were(29.42±1.76)%,(29.42±1.76)%,(29.42±0.74)%,(27.09±3.85)% and(25.09±3.89)%; the proliferation inhibition ratios of rh IL-29A143 P on the above tumor cells were(25.20±3.30)%,(23.84±2.93)%,(28.57±0.80)%,(25.96±1.41)% and(27.86±1.63)%. It indicate that make mutant on the amino acid residues of h IL-29 receptor binding domain may enhance its antitumor proliferation activity, which lay a foundation for develop the antitumor drugs of h IL-29.