Cloning And Expression Of E.coli Preferred Coding Sequences Of Human Interferon-γ Gene

Interferon-γ, also called Immune Interferon, is a kind of protein for function regulating, secreted by lymphocyte. Besides the function of antivirous and antitumor, it is still a kind of double-way immunomodulation factor in vivo with the potentiality in treating self-immunity disease. Because of the shortage of native human interferon-γ, scientists pay much attention to enhancing the expression level of recombinant human interferon-γ with the method of gene engineering.In this report, the codons in the native human interferon-γ cDNA were replaced by those E.coli preferred while maintaining the amino acid sequence. The modified coding sequence was divided into six fragments and synthesized each chemically in vitro. These fragments were connected with a series of PCR, and the final product was inserted into the expression vector pBV220. And then, the recombinant plasmid was transformed into E.coli JM109 for expression. SDS-PAGE and Western Blotting showed that the objective protein's molecular weight was about 18KD. The production of rhIFN-γ could account for 39.9% of the total proteins in E.coli JM109 with optimizing the culture conditions. The recombinant protein mainly existed in inclusion bodies. After solubilization and extraction, rhIFN-γ was renaturated by stepwise diluting and was dialyzed. The titer of rhIFN-γ's bioactivity was 5.6 X 10~3 IU/ml, and the specific activity was 2.8 ×10~4IU/mg.