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

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 cDN...

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Bibliographic Details
Published inScience in China. Series B, Chemistry, life sciences & earth sciences Vol. 37; no. 9; p. 1073
Main Authors Zhao, C W, Wang, J X, Xiao, D H, Ma, X K
Format Journal Article
LanguageEnglish
Published China 01.09.1994
Subjects
Animals
Baculoviridae - genetics
Base Sequence
Cell Line
Cloning, Molecular
DNA, Complementary - genetics
Gene Transfer Techniques
Interleukin-6 - biosynthesis
Interleukin-6 - genetics
Lepidoptera - cytology
Lepidoptera - genetics
Molecular Sequence Data
Plasmids
Polymerase Chain Reaction
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Summary: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.
ISSN:1001-652X