Quantitative PCR as a method for monitoring retroviral infection on the gene level.

For monitoring retroviral infection on the gene level, we propose the use of quantitative PCR with two internal standards: one for a fragment of the viral genome and the other for the host cell marker gene. The standards (one for HIV and the other for a human DNA marker gene HLA-DQ alpha) were constructed by PCR-mediated joining of DNA fragments and were found to be effective in quantitative PCR despite rather different structures of amplified fragments in target and standard DNAs. The number of HIV DNA copies was found to be 2-500 per 1000 lymphocytes in blood from HIV-infected patients and up to 5000+ per 1000 cells in chronically infected cell lines.

False-positive sera do not react with human immunodeficiency virus (HIV) gag-encoded recombinant antigen.

Ten sera from healthy blood donors positive by enzyme-linked immunoadsorbent assay (ELISA) were studied by immunoblot assay using natural and recombinant proteins. They interacted only with p17 or p24 proteins but were nonreactive with a recombinant protein (RP 50), which carries antigenic determinants to p17 and p24. Reactions were not blocked by preincubation of sera with genetically engineered p17 and p24 or purified viral p24, indicating that some new epitopes were formed during the Western blot procedure.

Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli.

A new expression vector (pBB1) has been constructed for the regulated expression of genes in Escherichia coli. Based on the pUC plasmids, the pBB1 carries lacIts allele of the lac repressor gene. This makes it possible to control expression of cloned genes by shifting the temperature from 30 degrees C to 42 degrees C.