Expression And Active Detection Of HIV-1Membrane Fusion Inhibitors HR212in Tobacco Plants

Human immunodeficiency virus (HIV) is the cause of the Acquired Immune Deficiency Syndrome(AIDS). HIV is belonged to enveloped virus. Only after membrane fusion can the genetic materials be released into the target cell. Fusion proteins of enveloped viruses promote viral infection by mediating the fusion of viral membrane with target cell membrane. According the structure of fusion proteins, we also class the fusion proteins into class I fusion proteins and class II fusion proteins.There are two heptad-repeat regions HR1peptides and HR2peptides in class I fusion proteins. As the fourth generation drugs against HIV, fusion inhibitors, with higher efficiency and lower toxicity, could impede the HIV’s invasion into cells at an early stage. The HR2and the HR1peptides are potent inhibitors of HIV-1entry.One of these peptides T.20was approved by the US food and drug administration(FDA)for treatment of HIV-1infection.Many similar inhibitors are being studied now. In such background, HR212was considered with good development prospect since its better properties than T20. But, the high cost would limit the universalness of the HR212if the production line relied on chemosynthesis.HR212’s codons and mRNA secondary structure was optimized via online database and prediction software. Nowadays many inhibitors of HIV-1is peptides. Because peptides have some disadvantage. So people hopes one kinds of drug that have no disadvantage as peptides can be found. On the other hand, in our study we also constructed two recombinant. Hoping HR212have activity in DNA form. In this study, we express and purify the inhibitor proteins of HIV-I:HR212(HR2-HR1-HR2).In this experiment, Firstly, we cloned HR212into the eucaryotic expressing vector pSPDK661and recombinant plasmids pSPDK661-HR212-GFP, and pSPDK661-HR212were constructed. The recombinant pSPDK661-HR212-GFP were transcribed and expressed in euearyotie cells effectively through the detecting of Western-blotting and cell exudation interdiction study. we chose tobacco plant to produce HR212protein. The codon optimized HR212gene was insert into plasmid pSPDK661, and the plant transformation vector(pSPDK661-HR212)was constructed to express the HR212protein. At the same time, the recombinant plasmid pSPDK661-HR212-GFP inhibited the HIV-1entry. By using the leaf-inserting,cultivated method,HR212gene was transferred into tobacco cell respectively. And these results indicated that the HR212was integrated into the tobacco genomic DNA. The western blotting result showed that the protein was expressed efficiently.This research provides a new method for new vaccine, which presents theorical significance and applicative prospect. According to the result, the studies provide fundamental for the further study on the new type DNA drug of HIV-1. Having realized the expression of HR212,This study has provided useful insights for the final goal of sectretory expression. Besides, the research has established a preliminary detecting method for HR212’s expression in future work.