Separation and purification of recombinant thymosin alpha 1 and interferon alfacon-1 fusion protein

Xianjun LIU; Fangxin LIU; Bo LI; Huiyun ZHOU

ABSTRACT

BACKGROUND:

It demonstrated that combined application of thymosin α 1 (TM-α1) and interferon (IFN) can enhance the antiviral activity of IFN.

OBJECTIVE:

To obtain recombinant fusion protein of TM- α1 and consensus IFN α (IFN α -con) with double activity of antiviral activity and immunity enhancement.DESIGN, TIME AND

SETTING:

The in vitro contrast experiment was conducted in the Biochemical Laboratory of Research Institute of Medicine, Chongqing K.E.W Pharmaceutical Co., Ltd. from March 2003 to December 2004.MATERIALS The fusion gene fragment (TM- α 1+IFN α -con) was synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, WISH cell, and vesicular stomatitis virus (VSV) was purchased from Institute of Biochemistry and Cell Biology, commercial products of IFN α 1b, IFN α 2a and TM- α 1 was served as reference substance.

METHODS:

The preference for E. coli of fusion sequence coding TM- α 1 and IFN α -con were cloned into expression vector of pET-22b(+) and expressed in BL21(DE3)-Codon plus-RP-X, which was purified by precipitation of (NH4)2SO4, hydrophobic interaction chromatography, anion-exchange chromatography, cation-exchange chromatography and molecular exclusion chromatography. The antiviral activity of fusion protein was tested by cytopathic-effect inhibition assay, and effect of fusion protein on lymphocyte proliferation was tested by cell proliferative assay.MAIN OUTCOME

MEASURES:

The specific activity of fusion protein and its biological activity in promoting lymphocyte proliferation.

RESULTS:

The fusion protein was expressed as a soluble form, accounting for over 20% of the total cell protein in E. coli, which approached 96% after purification. The antiviral activity of fusion protein was superior to IFN α 1b and IFN α 2a. However, the activity of fusion protein for promoting lymphocyte proliferation was similar to TM- α 1.

CONCLUSION:

The fusion protein of TM- α 1 and IFN α -con expressed in E. coli has both effects on anti-virus and promoting lymphocyte proliferation.