PROCESS FOR THE ISOLATION AND PURIFICATION OF HIGHER YIELD OF PROTEIN P17 OF HIV-1(SUBTYPE C)(A NON LIVING PROTEIN).

摘要

A simple and efficient method for hyper-expression in Escherichia coli and purification of matrix protein, p17, of human immunodeficiency virus type 1 (HTV-1) of both B- and C-subtypes is described. DNA sequences encoding p1 7. of B-and C-subtype were cloned from respective gag sequences. The gag sequences were obtained by PCR amplification using DNA extracted from peripheral blood lymphocytes of an HTV-1 infected patient from India. DNA sequence analysis revealed that die sample contained gag sequences of both HTV-1 B and C subtypes indicating dual infection. A T7 promoter based expression system was optimised for hyper-expression of pl7 iri the soluble form-Both pl7 (B- and C-subtype) were purified to near homogeneity using conventional chromatographic techniques. Purification of pl7 (C subtype) was described for the first time with yield of 7.7 mg from one litre culture. The yield of pl7 (B subtype) was 14.7 mg from one litre culture, which was several folds better than reported earlier. The irnmuno-reactivity of both types of pl7 to sera from HTV infected individuals was comparable. The method described here provides large quantities of highly pure pl7 of both B and C subtype, which can be used for structural, biochemical and immunologic^ characterization and, eventually for diagnostic and prognostic applications.