HIV-1 activity detection method to establish integrated and applied research

　　Title: HIV-1 activity detection method to establish integrated and applied research
　　Author: He Hongqiu
　　Degree-granting units: Beijing University
　　Keywords: human immunodeficiency virus (HIV);; integrase;; activity detection methods;; inhibitor screening;; protein activity and solubility
　　Abstract:
　　Human immunodeficiency virus (HIV) causes Acquired Immune Deficiency Syndrome (AIDS) is the most serious human history, the most deadly diseases. AIDS is still incurable. HIV-encoded integrase (IN)-mediated viral DNA integration with the host cell genome, viral replication is a key enzyme required for one. Inhibition of IN function can effectively block viral http://www.chinamagnets.biz/ replication in host cells. Also, because no normal human cells in the IN functional analogues, specific inhibitors acting on the IN side effects on the human body may be small. Therefore, IN is considered the ideal anti-HIV drug development targets.
　　In the body mainly through the catalytic IN 3 'processing and strand transfer reaction to achieve a two-step integration process. Above two-step reaction to in vitro using recombinant IN protein, oligonucleotide DNA substrate, and divalent metal ion-assisted implementation. In addition, IN catalytic chain transfer in vitro but also the reverse reaction - to integrate the chain transfer re-restored the formation of DNA products generated viral DNA and target DNA. IN establish an efficient activity detection method, and accordingly carry out the screening inhibitors, is currently IN inhibitors, the primary means of in vitro screening, but also to IN as a target for antiviral drug research and development of hot spots. In this study, expression and purification of recombinant IN protein, the establishment of the IN activity of a series of high-throughput detection methods, and application of these methods were IN the nature of inhibitor screening and other research. Content of the paper include the following aspects:
　　(1) integrase protein expression, purification and 3 'processing activity in high-throughput method for detection
　　Using PCR to obtain a HXB2CG strain IN gene by sequence analysis, mutations in primer design, using the overlapping PCR method HXB2CG IN mutations of HIV NL4-3 IN gene, and the introduction of mutations to improve protein solubility F185K/C280S. The target gene connected to the pET expression vector in E. coli BL21 to achieve efficient and soluble expression. By affinity chromatography from supernatants of cell disruption and purified to high purity with 3 'processing and strand transfer activity of recombinant IN protein function.
　　According to the principle of molecular beacons designed fluorophore and quencher labeled analog of viral DNA sequences 3 'processing of DNA substrates, we propose a test 3' processing of new reactive fluorescent analysis. Experiments show that the method is not only sensitivity, high specificity, simple operation, convenient for the whole liquid reaction environment, and real-time quantitative monitoring of IN 3 'processing reaction. This method can be used IN 3 'processing reaction and the properties of 3' processing of IN inhibitors that target high-throughput screening. This study established the 3 'processing reaction fluorescence detection method has the potential to be applied to other proteins and DNA interaction studies, has important academic and application value.
　　(2) integrase strand transfer activity of high-throughput method for detection and application
　　Inhibition of chain transfer is considered to be active in vivo inhibition of IN function key, the current IN inhibitors in vitro screening to chain transfer to the main target. This study were synthesized using biotin and digoxigenin-modified donor DNA and target DNA, the introduction of the streptavidin-biotin magnetic beads capture reaction DNA product, made a chain transfer activity of IN detection or detection of 3 'processing and the overall chain transfer activity of high-throughput enzyme-linked immunosorbent assay (ELISA).
　　Compared with the previous ways, our proposed high-throughput ELISA has significant improvements: (i) without the use of radioactive substrate to achieve high throughput; (ii) reaction, all reagents were suspended in a liquid environment free , the bead-DNA product of beads - easier access to more fully antibodies, significantly increased sensitivity, but also to respond flexibly applied to study the interaction of the various reagents and help study the mechanism of inhibitor; (iii) no coated, sealed microplate, time-saving, pre-test transfer beads to a new microplate to microplate almost completely eliminate non-specific adsorption of reagents, effectively reducing the background value and improve the specificity; (iv ) ELISA and fluorescent immunosorbent assay (FLISA) and two detection strategies used to increase the scope of application of the method.
　　Successful application of high-throughput ELISA studies of metal ions on the chain transfer reaction, obtained some interesting results. IN inhibitors with known proven high-throughput ELISA screening applied to the effectiveness of IN inhibitors, and extracts from natural products and synthetic compounds have successfully screened a number of preliminary activity of IN inhibitors.
　　(3) integration of the enzyme activity to integrate high-throughput method for detection
　　IN response to in vitro integration through to achieve a reversal of the integration process, and the integrase core area (IN-CCD) has an independent catalyst to integration. Established to integrate high-throughput detection method of the activity of IN function and screening IN inhibitors are of great significance. In this study, expression and purification of the functional activity of the wild-type IN and IN-CCD protein design and synthesis of biotin and digoxin to the integration of dual-labeled DNA substrate, based on the streptavidin-biotin magnetic beads to capture biotinylated DNA, established a testing and IN-CCD IN integration activity in vitro to high-throughput ELISA.
　　To study the integration of metal ions on the impact of high-throughput ELISA results showed that with 3 'processing and strand transfer reaction is similar, IN to the integration reaction is more preferred in the use of Mn2 + instead of Mg2 + ions for the metal-assisted. Further analysis showed that the use of NaCl titration experiments, IN to the integration of the preferences of Mn2 + and Mn2 + than Mg2 + more stable IN-DNA complexes related. Known IN inhibitors baicalein used to verify the integration of high-throughput ELISA screening the effectiveness of IN inhibitors, baicalein is clear proof to IN-CCD-targeting IN inhibitors. This study established to integrate high-throughput ELISA with a high-throughput, high sensitivity, high specificity, low background, time-saving, etc., can be applied to the disintegration reaction, the mechanism of inhibitors such studies, and has applied to the to IN for the target, especially with IN-CCD-targeted high-throughput screening of potential inhibitors.
　　(4) integration of the core area of ​​wild-type enzyme and F185K soluble mutant protein activity and solubility of
　　Wild-type (WT) IN and IN-CCD poor solubility in E. coli, are susceptible to the formation of inclusion bodies, purification and function of proteins to study the late inconvenience, and F185K mutations of IN and IN-CCD can achieve soluble expression and the activity is not Rare earth magnets affected. By construction, expression, purification of WT and F185K mutant IN-CCD, compared the differences in their solubility and activity. Analyzed and compared the WT and mutant IN proteins F185K/C280S solubility and activity differences. The results show that, F185K and F185K/C280S mutation of IN and IN-CCD significantly improve the solubility, to achieve soluble expression; same time, the mutation of IN-CCD to integrate the activity of a certain degree of reduction, IN 3 'processing and chain transfer activity to a certain extent reduced.
　　Further by homology modeling to construct a WT and F185K mutant IN-CCD protein structure, and in aqueous solution was 1800 ps molecular dynamics (MD) simulations, obtained some interesting results: (i) F185K mutations, the system features flexible loop region to some extent reduced, slightly reducing the overall movement of the catalytic DDE motif residues in the core of the distance between the no significant change, therefore, mutated protein catalytic activity, but activity was little impact; (ii) F185K mutation of IN-CCD electrostatic interactions within the network of protein conformational changes drive the changes, especially the loop region of significant conformational change, causing some of the surface proteins are embedded in hydrophobic residues, hydrophilic residues exposed, resulting in IN-CCD area relatively close to the polar solvent increases. Meanwhile, F185K mutant protein after the formation of hydrogen bonds between water has increased significantly, these changes make the IN-CCD solubility improved significantly. MD simulation and experimental results. The work is to understand the protein solubility and protein engineering of protein solubility transformation provides some valuable information and theoretical basis.
　　Degree Year: 2010