6th International AIDS Conference


San Francisco, California, USA — June 20-23, 1990

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Synthesis of the HTLV-1 p24 and gp46 proteins in eukaryotic cells using a baculovirus expression vector.

Int Conf AIDS 1990 Jun 20-23; 6:325 (abstract no. 1046)
McCreedy BJ, Earle SR; Organon Teknika Corp., Durham, North Carolina, USA

OBJECTIVE: Recombinant antigens produced in moderate yield and near native form are a potential alternative to whole viral lysate in diagnostic immunoassays. We have evaluated expression of the HTLV-1 core protein (p24) and external glycoprotein (gp46) in eukaryotic cells using a baculovirus expression vector.

METHODS: cDNA's containing the coding sequences for p24 and gp46 were synthesized from viral RNA templates. Plasmid clones were constructed containing the desired inserts. Following subcloning of the cDNA inserts into an appropriate plasmid transfer vector, the p24 and gp46 coding sequences were transferred to the genome of the baculovirus, AcMNPV, by cotransfection of insect cells (Spodoptera frugiperda) with wild type AcMNPV DNA. Recombinant baculoviruses containing the desired inserts were isolated and used to infect insect cells. Expression of recombinant p24 and gp46 proteins was confirmed by gel electrophoresis of infected cell lysates and immunoblotting using HTLV-1 reactive sera and monoclonal antibodies to HTLV-1 proteins.

RESULTS: Under the control of the viral polyhedrin promoter, high levels of recombinant p24 protein accumulated in infected cells by 48 hours post-infection. Analysis of the p24 produced in insect cells showed it to have a molecular weight and immunoblot reactivity identical to that of the native viral antigen. Similar reactivity was observed for the recombinant envelope glycoprotein. The recombinant gp46 was glycosylated and secreted into the culture medium confirming the ability of the insect cells to recognize and cleave signal sequences as well as carry out N-linked glycosylation.

CONCLUSIONS: Recombinant HTLV-1 p24 and gp46 proteins produced in insect cells closely resemble the native viral proteins in immunoreactivity. These recombinant antigens should prove useful in diagnostic assays.

Keywords: AEGIS, Baculoviridae, Human T-lymphotropic virus 1, Genetic Vectors, Recombinant Proteins, Viral Proteins, Spodoptera, Molecular Weight, Plasmids, Glycoproteins, Promoter Regions (Genetics), Immunoblotting, polyhedral protein, Animal, genetics, ICA6KWDaegis,baculoviridae,humant-lymphotropicvirus1,geneticvectors,recombinantproteins,viralproteins,spodoptera,molecularweight,plasmids,glycoproteins,promoterregions(genetics),immunoblotting,polyhedralprotein,animal,genetics,ica6

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