Establishment Of ELISA Assays For Detecting HIV-1 Integrase & Protease And Combining With Reverse Transcriptase Assay To Study HIV Multiple Targeting Inhibitors

AIDS is a pandemic infectious disease threatening the whole world including China. The 3 specific viral enzymes: reverse transcriptase, protease and integrase encoded by the HIV pol gene play key roles in HIV replication and are the rational targets for anti-HIV drugs. 10 HIV-reverse tracriptase inhibitors and 6 protease inhibitors have been approved for marketing and treatment of AIDS patients in clinic. Because these drugs are acting on one target of HIV replication, when used as monotherapy usually induced viral resistance rapidly and reduced the efficacy. Using combination of 2-4 anti-HIV drugs with different target to treat AIDS patients as "HAART"( Highly Active Antiretrovial Therapy) has made great success, but due to the serious toxicity, rebounding of virus titer after cessation of drug administration and expensive price of the drug, limited the application of present HIV treatment. To searching new kinds of effective anti-HIV drugs is a long-term and hard task..HIV-integrase encoded by 3' end of HIV pol gene integrated viral cDNA copy into the host chromosome and the viruses replicate with the infected cells is the main cause of the difficulty to clear off the virus. HIV integrase is not present in human cells, so it has been considered as an attractive therapeutic target for AIDS. But due to its poor solubility, only the 3 D structure of core domain was elucidated, computer based design of HIV integrase inhibiter is impossible, the ³²P labeled assay screening method is not fit for massive screening. Some HIV integrase inhibitors have been tested but not approved for clinical usage. For fascilitating the development HIV-integrase inhibitors, to set up a new ELISA assay will be beneficial. Using HIV integrase plasmid FI85K/C280S INI-288, a gift from Dr. Robert Craigie in NIH, USA was transformed into E. coli to express HIV integrase and purified by nickel-affinity chromatography, the isolated HIV integrase was identified by ³²P autoradiography, ELISA assay was established, it is quick and easily handled, the liability was confirmed by repeatedly evaluating known acive...