EXPRESSION OF HIV ANTIGENS IN E COLI

表达HIV病毒gag、env和pol基因部分的DNA克隆。

DNA clones expressing portions of the gag, env, and pol genes of HIV in E. coli have been isolated and characterized in order to derive information on antigenic epitopes and to analyze reactivities of human sera at different stages of virus infection. Eight clones expressing different regions of gag gene proteins pl5, p24, and p17 have been generated by recombinant DNA techniques. A ninth clone was prepared using the polymerase chain reaction followed by standard cloning techniques. These 9 clones were used to map epitopes on the gag gene recognized by a panel of monoclonal antibodies. A manuscript describing these studies has been published and an invention report filed. HIV integrase (IN) is a viral enzyme required for integration of virus DNA into the host chromosome. As part of a collaborative project with Dr. Judith Levin (NICHD/NIH) and Dr. Donald Court (FCRF) the pol clone pWS4-1 has been subcloned to express only the integrase (IN) gene in order to produce monospecific antisera. An IN clone was isolated which expresses a fusion protein containing 27 amino acids of the RT and all of IN and the protein was purified and used to immunize rabbits. The rabbit antisera are reactive with HIV-1 IN but not HIV-2 IN by Western blot, and also reactive with RT indicating that an antigenic epitope is present.in the terminal 27 amino acids of RT. An additional clone expressing only IN sequence has been constructed using PCR, and the protein can be isolated from gels in enzymatically active form. The recombinant protein and the assay we have developed may be useful in large-scale screening for HIV antivirals and an invention report has been filed. In addition, twenty-four hybridomas expressing mAbs against IN and RT have been prepared and are being subcloned and characterized.