Antiviral Active Ingredient Extraction And Separation Of The 1948 Fermentation Broth Of Streptomyces, Structure Determination And Antiviral Research

Screening for novel antiviral antibiotics from the secondery metabolites of microorganisms is an important strategy in the study of antiviral drugs. The screening model against specific targets can significantly improve the possibility of obtaining new compounds. This thesis has focused on three aspects of research work as follows:· The study on purification and characterization of HIV-1 reverse transcriptase from a strain of recombinant Escherichia coli.Among the HIV-1 proteins, reverse transcriptase( RT ) has been being a very useful target protein for the screening and design of specific inhibitors. Retrovirus replication is absolutely dependent on both the RNase H and the polymerase functions of RT and , so far as is now known, RT does not play a direct role in the life cycle of a normal cell. Under suitable fermentation conditions in our experiment, HIV-1 RT was highly expressed in E. coli JM 109 (pKRT-2) by inducing the trc promoter with isopropyl- [3 -D-thiogalactopyranoside(IPTG). 1.1 mg of purified Rtwas obtained from one liter culture of bacteria by DEAE-cellulose, phosphocellulose ,and sephadex G-100 chromatography. SDS-PAGE analysis of the purified RT showed two majer protein bands of 66KD and 51KD, indicating that the purified RT was a heterodimer composed of two subunits. Results of enzyme assay showed that the purified recombinant RT had high activity(1.4×10~4 unit/mg). We also improved the reaction system of enzyme assay. The effects of PFA on the recombinant HIV-1 RT were determined with the improved enzyme assay and the mechanism of inhibition was non -competetive with respect to substrate. This result is consistent with that of effects of PFA on native HIV-1 RT reported. This suggest that the purified recombinant HIV-1 RT has the same peoperties on the sensitivity to specific inhibitors and can be applied to the drug screening directly.