Molecular cloning and expression of human interleukin-6 in insect cells.

Abstract

670-bp hIL-6 cDNA fragments have been amplified by polymerase chain reaction (PCR) using recombinant plasmid pBMIL-6A as templates and two synthetic oligonucleotides containing the optimized translation initiation sequence and restriction sites suitable for cloning as primers. The amplified IL-6 cDNA fragments have then been recombined with a non-fusion expression baculovirus vector pVL1393. The resultant recombinant plasmid pVL. IL-6 together with wtAcMNPV DNAs were transferred into cultured lepidopteran insect cells (Sf9) by calcium phosphate coprecipitation procedure. The recombinant baculoviruses were formed by homologous recombination in vivo between pVL. IL-6 and wtAcMNPV DNAs, screened for plaque assay, and identified by means of dot blotting hybridization. The expressed rhIL-6 was secreted into the culture medium, and its bioactivity was measured through half-maximum H-TdR incorporation into IL-6-dependent cells 7TD1. As a result, the supernatant collected from recombinant baculovirus rAc. IL-6-infected Sf9 cells showed IL-6 activity of 10(6) U/mL. The expression level of rhIL-6 of the supernatant determined by IL-6 ELISA quantitation kit was 1 microgram/mL.