摘要
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 opti-mired translation initiation sequence/and restriction sites suitable for cloning as primers.The amplified IL-6cDNA fragments have then been recombined with a non-fusion expression baculovirus vector pVL1393.Theresultant recombinant plasmid pVL.IL-6 together with wtAcMNPV DNAs were transferred into culturedlepidopteran insect cells(Sf9)by calcium phosphate coprecipitation procedure.The recombinant baculovirus-es were formed by homologous recombination in vivo between pVL.IL-6 and wtAcMNPV DNAs,screenedfor plaque assay,and identified by means of dot blotting hybridization.The expressed rhIL-6 was secretedinto the culture medium,and its bioactivity was measured through half-maximum H-TdR incorporation intoIL-6-dependent cells 7TD1.As a result,the supernatant collected from recombinant baculovirus rAc.IL-6-infected Sf9 cells showed IL-6 activity of 10~6U/mL.The expression level of rhIL-6 of the supernatant deter-mined by IL-6 ELISA quantitation kit was 1 μg/mL.
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 opti- mired 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 baculovirus- es 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 10~6U/mL.The expression level of rhIL-6 of the supernatant deter- mined by IL-6 ELISA quantitation kit was 1 μg/mL.
基金
the National Natural Science Foundation of China
