[Construction and expression of huGM-CSF (9-127)-IL-6(29-184) fusion protein gene]

To construct and express huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities.

The novel gene coding for the fusion protein of huGM-CSF(9-127)-IL-6(29-184) was constructed by strategy of step by step cloning in pBV220 expression vector. The amino acids 1-8 of huGM-CSF and the amino acids 1-28 of huIL-6 were deleted by PCR technique. The mutant huGM-CSF (9-127) and huIL-6 (29-184) cDNAs were linked via a linker sequence coding 15 amino acid residues (G-G-S-G-S)3. Fusion protein was expressed in E.coli host strain DH5 alpha. To obtain the fusion protein, Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration were performed. The biologic activities were detected by MTT method.

Fusion protein was expressed in E.coli host strain DH5 alpha in the form of inclusion body. The expression level was more than 25% of the total cell lysate. Through Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration, huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity was obtained. The protein showed both huGM-CSF and huIL-6 biologic activities. The specific activity of huGM-CSF was 1.08 x 10(8) U/mg, and for huIL-6, it reached 1.95 x 10(7) U/mg.

huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities was obtained.