| Integrase (IN) of human immunodeficiency virus type I (HIV-1) is one of the three enzymes encoded by HIV-1 pol gene. The other two enzymes are reverse transcriptase (RT) and protease (PR). IN is necessary for the integration of viral cDNA into the chromosomal DNA of host cell, reverse transcription and nuclear import of viral pre-integration complex (PIC). The classic anti-retroviral agents target RT, PR. Because of the emergence of drug resistant HIV-1 strains, patients do not respond well to these drugs. Drug related toxicity (such as metablic disorders) limits their clinical application. More effective and safer anti-viral agents are expected. Because of the critical role of IN in HIV-1 replication, IN and its interaction with host cellular factors are attractive targets for the design of novel drugs against AIDS. The studies of IN C terminal domain (CTD) revealed that it plays special roles in the steps of HIV-1 replication. IN-CTD binds to DNA non-specifically, interacts with importin 7 and promotes the reverse transcription of viral RNA and the integration of viral cDNA. Current researches on IN are mostly involve mutational analysis, but this method is likely to change the comformation of IN. Using monoclonal antibodies (mAbs) to study the structure and function of IN proved to be advantageous, for it maintains the integrity of IN. Some studies illustrate that IN CTD interacts with host cellular importin7, but there is no report shows the particular domain of importin 7 which interacts with IN. In this study we prepared a panel of mAbs against IN by the aid of hybridoma technique; we studied the interaction between IN and importin 7 using cell-based co-immunoprecipitation (co-IP). We think that the knowledge of the structure, function, interaction with cellular factors of IN will provide a clue for developing novel agents and vaccine against HIV-1.In the first part of this study, we constructed an IN 142-288 amino acid fusion protein expression plasmid, pGEX-4T1-GST- IN142-288, which can express in a prokaryotic system efficiently. It was transformed into Escherichia coli BL 21 (DE3) and the fusion protein GST-IN 142-288 was purified. The other two fusion proteins of wild type IN, His-IN and T7-IN, were also purified as the antigen for screening hybridoma clones. BALB/c mice were immunized with purified IN 142-288 protein and the mAbs were developed by the aid of hybridoma technique. The mAbs were tested using enzyme-linked immuno-sorbent Assay, (ELISA), Western-blot (WB), Immuno-precipitation (IP) and immuno-fluorescence stain. 45 ELISA positive clones were obtained. Among them, there were 12 clones showing WB and IP positive and 3 clones showing immuno-fluorescence stain positive. Isotyping was performed to the 12 WB and IP positive clones. According to heavy-chain isotype, there were 3 IgG1, 6 IgG2a, 2 IgG2b and 1 IgM. According to light-chain isotype, all the clones wereκ.In the second part of this study, the interaction of importin 7 and IN was investigated. The plasmids of sVCMVin-T7-Imp7 207-836 and sVCMVin-T7-Imp7 442-836 were constructed, which expressed T7-importin 7 207-836 amino acid and T7-importin 7 442-836 amino acid fusion protein. The two importin 7 plasmids were separately co-transfected into HEK 293T cells with CMV-YFP-IN which expressed wide type IN. The interaction between importin7 fusion protein and IN fusion protein was analyzed using cell-base co-immuno-precipitation (co-IP). The results showed that T7-importin7 207-836 and T7-importin7 442-836 interacted with YFP-IN, indicating the binding domain may lie in the middle part of importin 7 and at least one binding domain is located in the 442-836 amino acid region.
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