Study On The Preparation Of Recombinant HNP_1,3 And The Inhibition Against HIV-1, HSV-1 In Vitro

Acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) infection have been a major public health problem worldwide for the past 20 years. According to the UNAIDS and WHO, it is estimated that more than sixty million people in the world are infected with HIV and the virus is spreading quickly in many countries, so it is urgent to get the control of the rapid expansion. Although highly active antiretroviral therapy (HAART) has led to profound and prolonged reductions in virus levels in many individuals, high cost, severe side effects and increasing emergence of multidrug-resistant virus strains, especially the partial clearance of latent virus limit the use of HAART. All these reasons necessitate continuation of the search for new drugs with high efficiency, low cytotoxicity and multi-targets.Human neutrophil peptide (HNP), or generally named alpha defensins are small cationic antimicrobial peptides predominantly found in azurophile of human polymorphonuclear cells. The ability to inactivate a broad range of microorganism including human immunodeficiency virus type I (HIV-1) particles render it a valuable drug in the search for anti-HIV-1 inhibitors, therefore much research is needed for the interaction of HNP and HIV-1 to elucidate the targets. In addition, the studies for HNP₁-3 against HIV are restricted to the limited sources, high cost and the low activity of purified or synthesized defensins, whilegene engineering provided a possible approach for production.In this study, with HNP13 cDNA cloned into eukaryotic expression vector, recombinant plasmids were cotransfected with plasmid pDCHlPll into CHO-dhfr' cells for gene amplification expression with methotraxate (MTX). HNPj 3 were harvested and confirmed with high biological activity. HIV-1 reverse transcriptase(RT) activity was tested for anti-HIV-1 infection when HNP13 interacted with virus at different steps in vitro to clarify the targets of inhibition against HIV-l;moreover, the antiviral activity against herpes simplex virus type I (HSV-l)was evaluated with the recombinant proteins. The research provides a basis for developing antiviral drugs on AIDS patients with complications. Works involving in this research are listed below:1. With RNA extracted from human neutrophil cells as template, cDNA encoding mature HNP13 was amplified by RT-PCR and then inserted into cloning vector pMD18-T. After restriction endonuclease digestion and DNA sequencing confirmation the gene was subcloned into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO. The gene encoding HNPj and HNP3 were cloned whose DNA sequence were 100% identical with that published in GenBank, then recombinant expression plasmids pcDNA3.1/V5-His-TOPO/HNPi,3 were successfully constructed. The successful construction of pcDNA3.1/V5-His-TOPO/HNPi,3 lays basis for the stable expression in mammalian cells.2. pcDNA3.1/V5-His-TOPO-HNPi and pcDNA3.1/V5-His-TOPO-HNP3 were cotransfected with pDCHlPll into CHO-dhfr' respectively for transient expression, and then selective medium containing G418 and MTX was performed for gene ampliation. High expression level was confirmed by ELISA; Expected DNA segments were amplified from stablly tranfectant clones by RT-PCR; Strong fluorescence was visible in cell plasma around the nuclear in the stablly transfectant cells by IFA; K-B disc agar diffusion test showed obvious bacteriastatic diffusions on MH plate of E.coli. The research provides a promising approach for biological production of defensins.3. With CD4 cells as target cells of HIV-1 acute infection, HNPi;3 at various...