VECTOR-DERIVED EXPRESSION OF RECOMBINANT RAT INTERLEUKIN-6

Rat interleukin-6 (IL-6) cDNA coding for an important inflammation- an d immune-regulatory polypeptide cytokine, was cloned into the novel ex pression vector pH6EX3 which directs the synthesis of inserted genes a s a fusion protein with histidine hexapeptide (HH), The resultant vect or (pRIL6.992) was shown to produce significant amounts of recombinant rat IL-6 fusion protein with HH at its N-terminus in various strains of Escherichia coil. The expression of the HH-IL-6 fusion protein was demonstrated to be under the control of the tac promoter and could be induced by IPTG. This protein was isolated from bacterial inclusion bo dies and purified to homogeneity in a one-step procedure by affinity c hromatography using a nickel-chelating column. The HH-IL-6 fusion prot ein isolated in this manner was biologically active as determined by h epatocyte stimulation and B9 hybridoma growth assays. Further, this ac tivity was neutralized with a polyclonal antiserum raised against rat IL-6 protein generated in a novel fashion from rabbits infected with a recombinant human type-5 adenovirus vector expressing rat IL 6 protei n (Ad5E3rIL6). The recombinant HH-IL-6 protein was then used to boost Ad5E3rIL6-immunized rabbits. This resulting antiserum was shown to neu tralize recombinant and natural rat and murine IL-6 bioactivity in vit ro and was useful in Western blot analysis and immunohistochemistry of rat IL-6. (C) 1996 Academic Press, Inc.