[Expression of soluble 55 kD receptor for human tumor necrosis factor using baculovirus vector].
Total RNA was isolated from HeLa cells and about 530 bp cDNA encoding shTNFR55 was amplified with RT-PCR from the total RNA. The cDNA was cloned into the downstream of polyhedrin gene promoter in transfer vector pAcGP67B, and a recombinant transfer plasmid pAcTNFR was constructed. Insect cell sf9 was co-transferred with recombinant plasmid pAcTNFR and baculovirus AcNPV DNA. Recombinant virus AcNPV-TNFR containing shTNFR55 gene were formed by homologous recombination in insect cells. They were purified by plaque assay and were confirmed by DNA dot blot. The biological activity of expressed product was determined with culturing TNF-susceptible L929 cell line, and the result showed that the expressed product could neutralize the cytotoxicity of TNF on L929 cell line. Ligand blot analysis revealed the expressed protein three discrete bands between 20-25 kD.
Wu
N
N
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100050.
Li
Y
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007313:Insecta / Q000166:cytology; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins