SUBSTRATE FOR DETERMINATION OF HUMAN IMMUNODEFICIENCY VIRUS INTEGRASE ACTIVITY Russian patent published in 2004 - IPC

FIELD: biochemistry, virology, medicine.

SUBSTANCE: substrate is designated for determination of activity of integrase of human immunodeficiency virus in vitro. Substrate is prepared by synthesis of oligonucleotide primers that are complementary to conservative region with the most similarity of genomes of different strains of human immunodeficiency virus and amplification of DNA sequence that is complementary to covalently closed ring forms of DNA of immunodeficiency virus, and by polymerase chain reaction. Then DNA product is prepared by repeated amplification with pair of nest primers and DNA product consists of two parallel oriented intact LTR-sequences with length 1200-1300 nucleotide pairs. Then prepared DNA product is labeled with Klenow's fragment and [α-³²P]-TTP for determination of activity of integrase in the 3'-processing reaction and using Klenow's fragment and a set of [α-³²P]-TTP for determination of activity of integrase in the insertion reaction.. The labeled DNA-product is used for determination of activity of integrase in vitro and the following analysis of products reaction in polyacrylamide gel and radioautography. The proposed substrate elevates the rate of determination of integrase activity in vitro in retention of specificity and identity of constructed substrate with natural substrate for integrase of human immunodeficiency virus.

EFFECT: improved assay method, valuable properties of substrate.

3 cl, 3 dwg, 5 ex