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 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.
Zhao
C W
CW
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing, PRC.
Wang
J X
JX
Xiao
D H
DH
Ma
X K
XK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018014:Gene Transfer Techniques; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics*; D007915:Lepidoptera / Q000166:cytology* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary; D015850:Interleukin-6