Expression, Purification Of HIV-1 Tat Protein And Preparation, Preliminary Application Of Its ScFv

The molecular biology research indicated HIV-1 is major pathogen for AIDS as epidemic in the world. The Tat protein of human immunodeficiency virus type 1 is a powerful transcriptional activator of viral gene expression and enhances the rate of human immunodeficiency virus type 1 long terminal repeat–specific transcription.In addition to this critical role as a transcription factor, a growing body of evidence suggests that Tat plays a role as an extracellular protein.Tat protein and its antibody were prepared in this research.The first extron of tat gene of HIV一1 B'subtype and the second extron of tat gene of HIV一1 B' subtype were fused as target gene, and the synthetic gene were replaced with the prefered codons of E.coli , The target gene was cloned in the gene of HisTag,Trx Tag and S·Tag in pET32a(+) vector.Then the fusion protein was expressed in prokaryotic system.And was lysed by sonication.identified by SDS-PAGE electrophoresis,the result showed the approximate 28 kD fusion protein was expressed eficiently in prokaryotic system,and the percentage of protein expression of the fusion protein Tat the total soluble protein was 30%. The summarized procedure of purification is as followed:Octyl-Fast Flow-cu-Chelating-Fast Flow --G25 desalinization --Q sepharose high performance chromatography.By this way,About 3.3mg fusion protein Tat was obtain from 1L culture ,the purity was about 95%.To obtain humanize anti-HIV-Tat single-chain antibodies(ScFv) from Tomlinson (I + J) phage antibody library. Panning of phage antibody library against HIV-Tat was conducted to select specific anti-HIV-Tat ScFv. The antigen binding activity and the variable genes of the selected antibodies were analyzed and identified. After the panning, a Positive clone ScFv-J-C4 were obtained, which could bind Tat specifically. DNA sequence analyses indicated that the variable region gene of the obtained belonged to the subgroups of immunoglobulin gene family. By-passing immunization, the specific human anti-Tat ScFv was prepared by using phage antibody library.Western blotting results showed that single-chain antibody(ScFv) could specifically bind to Tat .Immunohistochemistry assay showed that Tat protein could transform into cells.So the validity and specificity of single-chain antibodies(ScFv) being used in the detection of Tat protein .This research build a foundation for the further study of the function and application of synthetic ScFv against Tat.